Flavivirus detection methods employing recombinant antigens comprising a Japanese encephalitis (JEV) signal sequence

ABSTRACT

The present invention encompasses isolated nucleic acids containing transcriptional units which encode a signal sequence of one flavivirus and an immunogenic flavivirus antigen of a second flavivirus. The invention further encompasses a nucleic acid and protein vaccine and the use of the vaccine to immunize a subject against flavivirus infection. The invention also provides antigens encoded by nucleic acids of the invention, antibodies elicited in response to the antigens and use of the antigens and/or antibodies in detecting flavivirus or diagnosing flavivirus infection.

This is a divisional of U.S. patent application Ser. No. 09/826,115,filed Apr. 4, 2001 and issued as U.S. Pat. No. 7,227,011 on Jun. 5,2007, which is a continuation-in-part of, and claims priority to U.S.application Ser. No. 09/701,536, filed Jun. 18, 2001 now U.S. Pat. No.7,417,136, which is a national stage of international application No.PCT/US99/12298, filed Jun. 3, 1999, which claims the benefit of U.S.provisional application No. 60/087,908, filed Jun. 4, 1998. All of thelisted applications are incorporated herein in their entirety byreference.

FIELD OF THE INVENTION

This invention relates to novel vaccines, diagnostics and methods ofusing both in the treatment and prevention of the diseases caused byflaviviruses. In particular, the vaccines are recombinant nucleic acidswhich contain genes for structural proteins of flaviviruses, such asJapanese encephalitis virus (JEV), West Nile virus (WNV) or relatedflaviviruses. These vaccines serve as a transcriptional unit for thebiosynthesis of the virus protein antigens when administered in vivo.The diagnostics are compositions containing antigens produced from therecombinant nucleic acids that can be used to detect flavivirusinfection.

BACKGROUND OF THE INVENTION

Flaviviruses are members of the genus Flavivirus, which is classifiedwithin the family Flaviviridae. The flaviviruses are largely pathogenicto humans and other mammals. Flaviviruses that inflict disease uponhumans and animals include Alfuy, Apoi, Aroa, Bagaza, Banzi, Batu Cave,Bouboui, Bukalasa bat, Bussuquara, Cacipacore, Carey Island, CowboneRidge, Dakar bat, Dengue (serotypes 1, 2, 3 and 4), Edge Hill, Entebbebat, Gadgets Gully, Iguape, Ilheus, Israel turkey meningoencephalitis,Japanese encephalitis, Jugra, Jutiapa, Kadam, Karshi, Kedougou,Kokobera, Koutango, Kunjin, Kyasanur Forest disease, Langat, Meaban,Modoc, Montana myotis leukoencephalitis, Murray Valley encephalitis,Naranjal, Negishi, Ntaya, Omsk hemorrhagic fever, Phnom Penh bat,Potiskum, Powassan, Rio Bravo, Rocio, Royal Farm, Russian spring summerencephalitis, Saboya, Sal Vieja, San Perlita, Saumarez Reef, Sepik,Sokuluk, Spondweni, St. Louis encephalitis, Stratford, Tick-borneencephalitis—central European subtype, Tick-borne encephalititis—fareastern subtype, Tembusu, THCAr, Tyuleniy, Uganda S, Usutu, West Nile,Yaounde, Yellow fever, Yokose, Ziki, Cell fusing agent and other relatedflaviviruses, as listed in Kuno et al. (J. Virol. 72: 73-83 (1998)).

The flaviviruses contain the following three structural proteins: prM/M,the premembrane and membrane protein; E, the envelope protein; and C,the capsid protein. (Monath, in Virology (Fields, ed.), Raven Press, NewYork, 1990, pp. 763-814; Heinz and Roehrig, in Immunochemistry ofViruses II: The Basis for Serodiagnosis and Vaccines (van Regenmorteland Neurath, eds.), Elsevier, Amsterdam, 1990, pp. 289-305). M has amolecular weight (MW) of about 7-8 kilodaltons (kDa) and E has a MW ofabout 55-60 kDa. M is synthesized as a larger precursor termed prM. Thepr portion of prM is removed when prM is processed to form M protein inmature virions. M and E are located in the membrane of the flavivirusparticle, and so have long been considered to constitute importantimmunogenic components of the viruses.

The flaviviruses are RNA viruses comprising single stranded RNA having alength, among the various species, of about 10 kilobases (kb). The Cprotein, with a MW of 12-14 kDa, complexes with the RNA to form anucleocapsid complex. Several nonstructural proteins are also encoded bythe RNA genome which are termed NS1, NS2A, NS2B, NS3, NS4A, NS4B andNS5. The genome is translated within the host cell as a polyprotein,then processed co- or post-translationally into the individual geneproducts by viral- or host-specific proteases (FIG. 1).

The nucleotide sequences of the genomes of several flaviviruses areknown, as summarized in U.S. Pat. No. 5,494,671. That for JEV isprovided by Sumiyoshi et al. (Virology 161: 497-510 (1987)) andHashimoto et al. (Virus Genes 1: 305-317 (1988)). The nucleotidesequences of the virulent strain SA-14 of JEV and the attenuated strainSA-14-14-2, used as a vaccine in the People's Republic of China, arecompared in the work of Nitayaphan et al. (Virology 177: 541-552(1990)).

Nucleotide sequences encoding the structural proteins of otherflavivirus species are also known. In many cases, the sequences for thecomplete genomes have been reported. The sequences available includedengue serotype 1 virus, dengue serotype 2 virus (Deubel et al.,Virology 155: 365-377 (1986); Gruenberg et al., J. Gen. Virol. 69:1391-1398 (1988); Hahn et al. Virology 162: 167-180 (1988)), dengueserotype 3 virus (Osatomi et al., Virus Genes 2: 99-108 (1988)), dengueserotype 4 virus (Mackow et al., Virology 159: 217-228 (1987), Zhao etal., Virology 155: 77-88 (1986)), West Nile virus (Lanciotti et al.,Science 286: 2331-2333 (1999)), Powassan virus (Mandl et al., Virology194: 173-184 (1993)) and yellow fever virus (YFV) (Rice et al., Science229: 726-733 (1985)).

Many flaviviruses, including St. Louis encephalitis virus (SLEV), WNVand JEV, are transmitted to humans and other host animals by mosquitoes.They therefore occur over widespread areas and their transmission is noteasily interrupted or prevented.

West Nile fever is a mosquito-borne flaviviral infection that istransmitted to vertebrates primarily by various species of Culexmosquitoes. Like other members of the Japanese encephalitis (JE)antigenic complex of flaviviruses, including JE, SLE and Murray Valleyencephalitis (MVE) viruses, WNV is maintained in a natural cycle betweenarthropod vectors and birds. The virus was first isolated from a febrilehuman in the West Nile district of Uganda in 1937 (Smithburn et al., Am.J. Trop. Med. Hyg. 20: 471-492 (1940)). It was soon recognized as one ofthe most widely distributed flaviviruses, with its geographic rangeincluding Africa, the Middle East, Western Asia, Europe and Australia(Hubalek et al., Emerg. Infect. Dis. 5: 643-50 (1999)). Clinically, WestNile fever in humans is a self-limited acute febrile illness accompaniedby headache, myalgia, polyarthropathy, rash and lymphadenopathy (Monathand Tsai, in Clinical Virology, (Richman, Whitley and Hayden eds.),Churchill-Livingtone, New York, 1997, pp. 1133-1186). Acute hepatitis orpancreatis has been reported on occasion and cases of WNV infection inelderly patients are sometimes complicated by encephalitis or meningitis(Asnis et al., Clin. Infect. Dis. 30: 413-418 (2000)). Thus, infectionby WNV is a serious health concern in many regions of the world.

The geographical spread of the disease, particularly the introduction ofWNV into the U.S. in 1999, has greatly increased awareness of the humanand animal health concerns of this disease. Between late August andearly September 1999, New York City and surrounding areas experienced anoutbreak of viral encephalitis, with 62 confirmed cases, resulting inseven deaths. Concurrent with this outbreak, local health officialsobserved increased mortality among birds (especially crows) and horses.The outbreak was subsequently shown to be caused by WNV, based onmonoclonal antibody (Mab) mapping and detection of genomic sequences inhuman, avian and mosquito specimens (Anderson et al., Science 286:2331-2333 (1999); Jia et al., Lancet 354: 1971-1972 (1999); Lanciotti etal., Science 286: 2333-2337 (1999)). Virus activity detected during theensuing winter months indicated that the virus had established itself inNorth America (Morb. Mortal. Wkly. Rep. 49: 178-179 (2000); Asnis etal., Clin. Infect. Dis. 30: 413-418 (2000); Garmendia et al., J. Clin.Micro. 38: 3110-3111 (2000)). Surveillance data reported from thenortheastern and mid-Atlantic states during the year 2000 confirmed anintensified epizootic/epidemic transmission and a geographic expansionof the virus with documentation of numerous cases of infection in birds,mosquitoes and horses, as well as cases in humans (Morb. Mortal. Wkly.Rep. 49: 820-822 (2000)).

Currently, no human or veterinary vaccine is available to prevent WNVinfection and mosquito control is the only practical strategy to combatthe spread of the disease.

Japanese encephalitis virus (JEV) infects adults and children and thereis a high mortality rate among infants, children and the elderly inareas of tropical and subtropical Asia (Tsai et al., in Vaccines(Plotkin, ed.) W. B. Saunders, Philadelphia, Pa., 1999, pp. 672-710).Among survivors, there are serious neurological consequences, related tothe symptoms of encephalitis, that persist after infection. In moredeveloped countries of this region, such as Japan, the Republic of China(Taiwan) and Korea, JEV has been largely controlled by use of a vaccineof inactivated JEV. Nevertheless, it is still prevalent in othercountries of the region.

Vaccines available for use against JEV infection include live virusinactivated by such methods as formalin treatment, as well as attenuatedvirus (Tsai et al., in Vaccines (Plotkin, ed.) W. B. Saunders,Philadelphia, Pa., 1994, pp. 671-713). Whole virus vaccines, althougheffective, do have certain problems and/or disadvantages. The virusesare cultivated in mouse brain or in cell culture using mammalian cellsas the host. Such culture methods are cumbersome and expensive.Furthermore, there is the attendant risk of incorporating antigens fromthe host cells, i.e., the brain or other host, into the final vaccineproduct, potentially leading to unintended and undesired allergicresponses in the vaccine recipients. There is also the risk ofinadvertent infection among workers involved in vaccine production.Finally, there is the risk that the virus may not be fully or completelyinactivated or attenuated and thus, the vaccine may actually causedisease.

Dengue fever and dengue hemorrhagic fever (DF/DHF) are caused by denguevirus, which is also a mosquito-borne flavivirus. There are fourantigenically related, but distinct, dengue virus serotypes, (DEN-1,DEN-2, DEN-3 and DEN-4), all of which can cause DF/DHF. Symptoms of DF,the mild form of dengue-related disease, include fever, rash, severeheadache and joint pain. Mortality among those subjects suffering fromDF is low; however, among those subjects suffering from DHF, mortalitycan be as high as 5%. From available evidence, more than 3 million casesof DHF and 58,000 deaths have been attributed to DHF over the past 40years, making DHF a major emerging disease (Halstead, in Dengue andDengue Hemorrhagic Fever (Gubler and Kuno, eds.) CAB International, NewYork, N.Y., (1997) pp 23-44). Nevertheless, despite decades of effort,safe and effective vaccines to protect against dengue virus infectionare not yet available.

Yellow fever is prevalent in tropical regions of South America andsub-Saharan Africa and is transmitted by mosquitos. Infection leads tofever, chills, severe headache and other pains, anorexia, nausea andvomiting, with the emergence of jaundice. A live virus vaccine, 17D,grown in infected chicken embryos, is considered safe and effective.Nevertheless, there remains a need for a vaccine that is stable underadverse conditions, such as are commonly encountered in the tropicalregions of Africa and the Americas where the vaccine is most needed.

A recombinant flavivirus which is a chimera between two flaviviruses isdisclosed in PCT publication WO 93/06214. The chimera is a constructfusing non-structural proteins from one “type,” or serotype, of denguevirus or a flavivirus, with structural proteins from a different “type,”or serotype, of dengue virus or other flavivirus.

Several recombinant subunit and viral vaccines have been devised inrecent years. U.S. Pat. No. 4,810,492 describes the production of the Eglycoprotein of JEV for use as the antigen in a vaccine. Thecorresponding DNA is cloned into an expression system in order toexpress the antigen protein in a suitable host cell such as E. coli,yeast, or a higher organism cell culture. U.S. Pat. No. 5,229,293discloses recombinant baculovirus harboring the gene for JEV E protein.The virus is used to infect insect cells in culture such that the Eprotein is produced and recovered for use as a vaccine.

U.S. Pat. No. 5,021,347 discloses a recombinant vaccinia virus genomeinto which the gene for JEV E protein has been incorporated. The liverecombinant vaccinia virus is used as the vaccine to immunize againstJEV. Recombinant vaccinia viruses and baculoviruses in which the virusesincorporate a gene for a C-terminal truncation of the E protein ofdengue serotype 2, dengue serotype 4 and JEV are disclosed in U.S. Pat.No. 5,494,671. U.S. Pat. No. 5,514,375 discloses various recombinantvaccinia viruses which express portions of the JEV open reading frameextending from prM to NS2B. These pox viruses induced formation ofextracellular particles that contain the processed M protein and the Eprotein. Two recombinant viruses encoding these JEV proteins producedhigh titers of neutralizing and hemagglutinin-inhibiting antibodies, andprotective immunity, in mice. The extent of these effects was greaterafter two immunization treatments than after only one. Recombinantvaccinia virus containing genes for the prM/M and E proteins of JEVconferred protective immunity when administered to mice (Konishi et al.,Virology 180: 401-410 (1991)). HeLa cells infected with recombinantvaccinia virus bearing genes for prM and E from JEV were shown toproduce subviral particles (Konishi et al., Virology 188: 714-720(1992)). Dmitriev et al. reported immunization of mice with arecombinant vaccinia virus encoding structural and certain nonstructuralproteins from tick-borne encephalitis virus (J. Biotechnology 44: 97-103(1996)).

Recombinant virus vectors have also been prepared to serve as virusvaccines for dengue fever. Zhao et al. (J. Virol. 61: 4019-4022 (1987))prepared recombinant vaccinia virus bearing structural proteins and NS1from dengue serotype 4 and achieved expression after infecting mammaliancells with the recombinant virus. Similar expression was obtained usingrecombinant baculovirus to infect target insect cells (Zhang et al., J.Virol. 62: 3027-3031(1988)). Bray et al. (J. Virol. 63: 2853-2856(1989)) also reported a recombinant vaccinia dengue vaccine based on theE protein gene that confers protective immunity to mice against dengueencephalitis when challenged. Falgout et al. (J. Virol 63: 1852-1860(1989)) and Falgout et al. (J. Virol. 64: 4356-4363 (1990)) reportedsimilar results. Zhang et al. (J. Virol 62: 3027-3031 (1988)) showedthat recombinant baculovirus encoding dengue E and NS1 proteins likewiseprotected mice against dengue encephalitis when challenged. Othercombinations in which structural and nonstructural genes wereincorporated into recombinant virus vaccines failed to producesignificant immunity (Bray et al., J. Virol. 63: 2853-2856 (1989)).Also, monkeys failed to develop fully protective immunity to denguevirus challenge when immunized with recombinant baculovirus expressingthe E protein (Lai et al. (1990) pp. 119-124 in F. Brown, R. M.Chancock, H. S. Ginsberg and R. Lerner (eds.) Vaccines 90: Modernapproaches to new vaccines including prevention of AIDS, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

Immunization using recombinant DNA preparations has been reported forSLEV and dengue-2 virus, using weanling mice as the model (Phillpotts etal., Arch. Virol. 141: 743-749 (1996); Kochel et al., Vaccine 15:547-552 (1997)). Plasmid DNA encoding the prM and E genes of SLEVprovided partial protection against SLEV challenge with a single ordouble dose of DNA immunization. In these experiments, control miceexhibited about 25% survival and no protective antibody was detected inthe DNA-immunized mice (Phillpotts et al., Arch. Virol. 141: 743-749(1996)). In mice that received three intradermal injections ofrecombinant dengue-2 plasmid DNA containing prM, 100% developedanti-dengue-2 neutralizing antibodies and 92% of those receiving thecorresponding E gene likewise developed neutralizing antibodies (Kochelet al., Vaccine 15: 547-552 (1997)). Challenge experiments using atwo-dose schedule, however, failed to protect mice against lethaldengue-2 virus challenge.

The vaccines developed to date for immunizing against infection by JEV,SLEV, dengue virus and other flaviviruses have a number of disadvantagesand problems attending their use. Inactivated vaccine is costly andinconvenient to prepare. In addition, any such vaccine entails the riskof allergic reaction originating from proteins of the host cell used inpreparing the virus. Furthermore, such vaccines present considerablerisk to the workers employed in their production. Candidate attenuatedJEV vaccines are undergoing clinical trials, but as of 1996 have notfound wide acceptance outside of the People's Republic of China(Hennessy et al., Lancet 347: 1583-1586 (1996)).

Recombinant vaccines based on the use of only certain proteins offlaviviruses, such as JEV, produced by biosynthetic expression in cellculture with subsequent purification or treatment of antigens, do notinduce high antibody titers. Also, like the whole virus preparations,these vaccines carry the risk of adverse allergic reaction to antigensfrom the host or to the vector. Vaccine development against dengue virusand WNV is less advanced and such virus-based or recombinantprotein-based vaccines face problems similar to those alluded to above.

There is therefore a need for vaccines or improved vaccines directedagainst flaviviruses such as yellow fever virus, dengue virus, JEV, SLEVand WNV which are inexpensive to prepare, present little risk to workersinvolved in their manufacture, carry minimal risk of adverseimmunological reactions due to impurities or adventitious immunogeniccomponents and are highly effective in eliciting neutralizing antibodiesand protective immunity. There is furthermore a need for a vaccineagainst JEV, WNV and related flaviviruses that minimizes the number ofimmunizing doses required.

Many of the shortcomings of the current art as described in detail forthe production of vaccines also apply to the production of antigens andantibodies to be used for the production of immunodiagnostics.Particularly, the concurrent risks and costs involved in the productionof antigens from viruses and the failure of most currently availablerecombinantly expressed antigens to elicit effective immune responsesare paralleled in the field of immunodiagnostics by the same risks, highcosts and a corresponding lack of sensitivity. Thus, because of the highcosts, risk of accidental infection with live virus and the lower thandesired levels of sensitivity of the previously available tests, thereexists a need for rapid, simple and highly sensitive diagnostic testsfor detecting flavivirus infection and/or contamination.

The present invention meets these needs by providing highly immunogenicrecombinant antigens for use in diagnostic assays for the detection ofantibodies to selected flaviviruses. The present invention furtherprovides for the use of recombinant antigens derived from flaviviruses,flavivirus genes or mimetics thereof in immunodiagnostic assays for thedetection of antibodies to flavivirus proteins.

SUMMARY OF THE INVENTION

The present invention provides a nucleic acid molecule which contains atranscriptional unit (TU) for an immunogenic flavivirus antigen. The TUdirects a host cell, after being incorporated within the cell, tosynthesize the antigen. In an important aspect of the invention, theflavivirus can be yellow fever virus (YFV), dengue serotype 1 virus(DEN-1), dengue serotype 2 virus (DEN-2), dengue serotype 3 virus(DEN-3), dengue serotype 4 virus (DEN-4), St. Louis encephalitis virus(SLEV), Japanese encephalitis virus (JEV), West Nile virus (WNV),Powassan virus or any other flavivirus. In important embodiments of thepresent invention, the antigen can be the flavivirus prM/M protein, theE protein, or both. In particular, when the TU includes both the prM/Mand E proteins, the host cell secretes subviral particles containing theprM/M and E antigens. In a further important aspect of the invention,the nucleic acid is a DNA molecule. In additional significantembodiments, the nucleic acid TU includes a control sequence disposedappropriately such that it operably controls the expression of the prM/Mand E antigens and this control sequence can be the cytomegalovirusimmediate early promoter. In an additional embodiment, the nucleotidesequence of the TU is engineered to optimize eukaryotic translation byminimizing large hairpin structures in the 5′-end untranslated region ofan mRNA produced by the TU and/or the inclusion of a Kozak consensussequence at the translational start site of an mRNA produced by the TU.In an additional embodiment, the transcriptional unit also includes apoly-A terminator.

The present invention further provides a host cell comprising a nucleicacid molecule which includes a transcriptional unit for an immunogenicflavivirus antigen that directs the host cell to synthesize theimmunogenic antigen. The flavivirus may be YFV, DEN-1, DEN-2, DEN-3,DEN-4, SLEV, JEV, WNV, Powassan virus or other flavivirus. In importantembodiments, the antigen may be the prM/M protein, the E protein, orboth the prM/M and the E proteins. In the latter case, the cell secretessubviral particles containing the prM/M and E antigens.

Additionally, the invention provides a composition for vaccinating asubject against a flavivirus containing a nucleic acid molecule thatincludes a transcriptional unit for an immunogenic flaviviral antigen.The transcriptional unit directs a cell within the body of the subject,after being incorporated therein, to synthesize the immunogenic antigen.The composition further includes a pharmaceutically acceptable carrier.In significant embodiments, the flavivirus may be YFV, DEN-1, DEN-2,DEN-3, DEN-4, SLEV, JEV, WNV, Powassan virus or other flavivirus.Furthermore, the antigen may be the prM/M protein, the E protein, orboth the prM/M and the E proteins. In the latter instance, the cellsecretes subviral particles comprising the flavivirus prM/M and Eantigens. These subviral particles are also referred to as noninfectiousrecombinant antigen (NRA). In important embodiments, the nucleic acidmolecule is a DNA molecule. In further significant embodiments, thetranscriptional unit additionally contains a control sequence disposedappropriately such that it operably controls the synthesis of the prM/Mand E antigens when the nucleic acid is introduced into the cell of thesubject. This control sequence can be the cytomegalovirus immediateearly promoter. In a still further embodiment, the transcriptional unitcan also include a poly-A terminator.

The invention provides still further a method of immunizing a subjectagainst infection by a flavivirus. The method involves administering tothe subject an effective amount of a vaccinating composition thatcontains a nucleic acid molecule which includes a transcriptional unitfor an immunogenic flavivirus antigen. The transcriptional unit directsa cell within the body of the subject, after being taken up by the cell,to synthesize the immunogenic antigen. The composition additionallyincludes a pharmaceutically acceptable carrier. In significantembodiments of the method, the flavivirus may be YFV, DEN-1, DEN-2,DEN-3, DEN-4, SLEV, JEV, WNV, Powassan virus or other flavivirus. In yetother important aspects of the method, the antigen may be the prM/Mprotein, the E protein, or both the prM/M and the E proteins. When theantigen is both the prM/M and the E proteins, the cell within the bodyof the subject, after incorporating the nucleic acid within it, secretessubviral particles comprising the flaviviral prM/M and E antigens.Additionally, in significant embodiments of the method, the vaccinatingcomposition is administered to the subject in a single dose, via aparenteral route. In yet a further aspect of the method, the nucleicacid is a DNA molecule. In yet additional embodiments of the method, thetranscriptional unit further includes a control sequence disposedappropriately such that it operably controls the synthesis of the prM/Mand E antigens and in a significant aspect of this embodiment, thecontrol sequence is the cytomegalovirus immediate early promoter.Furthermore, the transcriptional unit may include a poly-A terminator.

These aspects and embodiments of the invention are the basis for itsdistinct attributes and advantages. Being a nucleic acid constructinvolving only portions of the flavivirus genome rather than thesequence encompassing the complete genome, the nucleic acidTU-containing vaccine is completely nonviable. It therefore poses nodanger of infection by the flavivirus to those involved in itsmanufacture or to subjects receiving the vaccine. The nucleic acidvaccine is easy to prepare and easy to administer and is stable instorage prior to use. Unexpectedly it has been found that the nucleicacid vaccine of the invention is essentially 100% successful inconferring protective immunity in mammals after administering only asingle dose. A further unexpected result is that the nucleic acid TU isable to engender immunity to a flavivirus in a female mammal which canbe transmitted to its progeny through the milk. Without wishing to belimited by theory, the inventor believes that a possible mechanism forthe success of the nucleic acid in conferring protective immunity isthat a host cell harboring the nucleic acid, such as the cell of asubject to whom the vaccine is administered, produces subviral particlescontaining the flaviviral prM/M and E antigens. These particles mimicthe immunogenic attributes of native flavivirus virions.

The present invention also provides noninfectious antigenicpolypeptides, antigenic polypeptide fragments and NRA comprising theprM/M and/or E proteins of flaviviruses, wherein the transmembranesignal sequence is derived from a first flavivirus and the M and/or Eproteins are derived from a second flavivirus. Further, the prM/Mprotein can comprise amino acid sequences from both the first and thesecond flaviviruses. “Chimeric” as used herein means any protein ornucleic acid comprising sequence from more than one flavivirus. As usedherein, “non-virulent” means the antigen or vaccine of this invention isincapable of causing disease. More particularly, the recombinant proteinantigens are free of contaminating genomic material from flavivirusesthat is necessary for flavivirus infection, replication andpathogenesis.

The polypeptides of the present invention can comprise the amino acidsequences defined herein, or that are known in the art, of the prM, Mand/or E proteins of selected flaviviruses. The nucleic acids of thisinvention can comprise nucleotide sequence that encodes the prM, Mand/or E proteins of selected flaviviruses.

The antigens of the present invention can be unconjugated, or they canbe conjugated to a carrier molecule that facilitates placement of theantigen on a solid phase. A carrier molecule is one to which antigenscan be conjugated and which will not react with antibodies in humanserum. An example of such a carrier is bovine serum albumin (BSA).

The antigens of the present invention can also be recombinant proteinsobtained by expressing nucleic acids encoding the antigen in anexpression system capable of producing the antigen.

The amino acid sequences of the present antigens can contain animmunoreactive portion of the prM, M and/or E antigen. These antigensmay further be attached to sequences designed to provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding to increase the reactivity of an epitope by providinga more rigid secondary structure, to increase its bio-longevity or toalter its cytotoxicity or to prevent infection. In any case, the antigenmust possess immunoreactivity and/or immunogenicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of flaviviral polyproteinprocessing. The central horizontal region provides a schematicrepresentation of the viral genome. The lines denote the 5′ and 3′non-translated regions and the boxed regions represent the open readingframe for structural (left and top) and non-structural (right andbottom) proteins. Cleavage by host cell signalase occurs simultaneouslywith translation at the E protein C-terminus, separating structural andnon-structural regions. A subtilase-like cellular enzyme, furin, may beresponsible for prM cleavage. Potential transmembrane domains of viralpolyprotein are indicated by shaded areas.

FIG. 2 is a map of the JEV genome (top) and the DNA sequence ofoligonucleotides used in a reverse transcriptase-polymerase chainreaction (RT-PCR) (center) to construct the transcription unit for theexpression of prM-E protein coding regions (bottom). Potentialtransmembrane domains of viral polyprotein are indicated by shadedareas.

FIG. 3 shows a schematic representation of the plasmid vectors, pCDNA3,pCBamp, and pCIBamp, and the relationship between them. These plasmidsinclude the CMV (cytomegalovirus) promoter/enhancer element, BGHp(A)(bovine growth hormone polyadenylation signal and transcriptiontermination sequence), ampicillin resistance gene and ColE1 origin ofreplication for selection and maintenance in E. coli. The f1 origin ofreplication for single-stranded rescue in E. coli cells, SV40 origin ofreplication (SV40 ORI), neomycin resistance coding region and SV40p(A)sequences were deleted from pCDNA3 to generate pCBamp. An intronsequence was inserted in the NcoI-KpnI site of pCBamp to generateplasmid pCIBamp.

FIG. 4 shows SDS-PAGE-immunoblot analyses of the sucrose gradientpurified subviral particles from JE-4B COS-1 culture fluid (4B, rightlane of each pair). The density gradient purified JE virion from JEVinfected C6/36 cell culture was used as a positive control (JEV, leftlane of each pair). JE HIAF (hyperimmune ascitic fluid); 4G2, anti-Emonoclonal antibody; JM01, anti-M monoclonal antibody; NMAF (normalmouse ascitic fluid).

FIG. 5 shows a profile of the E antigen in a rate zonal sucrose gradientanalysis prepared from the PEG precipitate of JE-4B cell culture mediumwith or without Triton X-100 treatment.

DETAILED DESCRIPTION OF THE INVENTION

The invention encompasses nucleic acid transcriptional units whichencode flaviviral antigenic proteins, such as the prM/M and E proteinantigens. The nucleic acids function to express the prM/M and E proteinantigens when the nucleic acid is taken up by an appropriate cell,especially when the cell is the cell of a subject. The invention alsoencompasses a vaccine whose active agent is the nucleic acidtranscriptional unit (TU). The invention further encompasses cellscontaining a TU. The invention in addition encompasses a method ofimmunizing a subject against flaviviral infection by administering tothe subject an effective amount of a vaccine containing the nucleic acidTU molecules.

The invention provides an isolated nucleic acid comprising atranscriptional unit encoding a signal sequence of a structural proteinof a first flavivirus and an immunogenic flavivirus antigen of a secondflavivirus, wherein the transcriptional unit directs the synthesis ofthe antigen. The invention further encompasses the use of the nucleicacid transcriptional unit (TU) to generate flaviviral antigens and theflaviviral antigens produced by the nucleic acid TU. The invention stillfurther encompasses the use of the flaviviral antigens encoded by the TUof the invention to produce flavivirus-specific antibodies and to detectthe presence of flavivirus-specific antibodies.

In one embodiment, the isolated nucleic acid of this invention cancomprise a transcriptional unit encoding a Japanese encephalitis virussignal sequence.

In another embodiment, the transcriptional unit of this invention canencode an immunogenic flavivirus antigen which can be from one or moreof the following flaviviruses: yellow fever virus, dengue serotype 1virus, dengue serotype 2 virus, dengue serotype 3 virus, dengue serotype4 virus, Japanese encephalitis virus, Powassan virus and West Nilevirus.

In a particular embodiment, the nucleic acid of this invention canencode a signal sequence of Japanese encephalitis virus and an M proteinand an E protein of West Nile virus, SLEV, YFV and/or Powassan virus.The nucleic acid can also encode an immunogenic antigen which can be anM protein of a flavivirus, an E protein of a flavivirus, both an Mprotein and an E protein of a flavivirus, a portion of an M protein of aflavivirus, a portion of an E protein of a flavivirus and/or both aportion of an M protein of a flavivirus and a portion of an E protein ofa flavivirus. In a preferred embodiment, the isolated nucleic acidencodes both the M protein and the E protein of the flavivirus. Further,the nucleic acid of the invention can be DNA and can comprise nucleotidesequence SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23.

The transcriptional unit of this invention can also comprise a controlsequence disposed appropriately so that it operably controls thesynthesis of the antigen. The control sequence can be, for example, thecytomegalovirus immediate early promoter. The nucleic acid of thisinvention can also comprise a Kozak consensus sequence located at atranslational start site for a polypeptide comprising the antigenencoded by the transcriptional unit. The transcriptional unit of thisinvention can also comprise a poly-A terminator.

The present invention further provides a cell comprising the nucleicacid of this invention.

Also provided is a composition comprising a pharmaceutically acceptablecarrier and nucleic acid or cell or antigen of this invention. Thepresent invention additionally provides a method of immunizing a subjectagainst infection by a flavivirus, comprising administering to thesubject an effective amount of a composition of this invention. In aparticular embodiment, the composition used to immunize a subjectdirects the synthesis of both the M protein and the E protein of aflavivirus and a cell within the body of the subject, afterincorporating the nucleic acid within it, secretes subviral particlescomprising the M protein and the E protein. Alternatively, thecomposition can comprise an M protein and/or E protein of a flavivirusor subviral particles comprising the M protein and E protein. In themethods of this invention, the immunizing composition can beadministered to the subject in a single dose and can be administered viaa parenteral route.

This invention further provides the antigens produced from the isolatednucleic acids of this invention. As an example, the antigen from thesecond flavivirus encoded by the nucleotide sequence of TU can be the Mprotein which can be, for example, from West Nile virus. The antigen canalso be protein from dengue virus, St. Louis encephalitis virus,Japanese encephalitis virus, Powassan virus and/or yellow fever virus.In a further embodiment, the antigen comprises a prM/M proteincomprising the transmembrane signal sequence from a first flavivirus andfurther amino acid sequence comprising the remainder of the prM/Mprotein from a second flavivirus, which can be from SLEV, JEV, YFV, WNVand/or Powassan virus.

The antigen encoded by the nucleotide sequence of the TU can be WestNile virus antigen, dengue virus antigen, St. Louis encephalitis virusantigen, Japanese encephalitis virus antigen, Powassan virus antigenand/or yellow fever virus antigen.

The antigen encoded by the nucleotide sequence of the TU can also be theE protein, which can be the E protein from West Nile virus, denguevirus, St. Louis encephalitis virus, Japanese encephalitis virus,Powassan virus and/or yellow fever virus.

Additionally, the antigen encoded by the nucleotide sequence of the TUcan be the M protein and the E protein, which can be from West Nilevirus, dengue virus, St. Louis encephalitis virus, Japanese encephalitisvirus, Powassan virus and/or yellow fever virus.

As used herein, “M protein” or “pr/M protein” or “prM/M protein” means aflavivirus M protein or flavivirus prM protein. Examples include, butare not limited to, prM proteins comprising amino acid sequence from oneor more flavivirus prM proteins, M proteins comprising no additionalamino acid sequence and proteins comprising additional amino acidsequences which are processed in vitro or in vivo to generate the matureM protein.

As used herein, “nucleic acid transcriptional unit” or “nucleic acidtranscriptional unit molecule” means a nucleic acid encoding one or morespecified proteins. The TU has biological activity such that, afterhaving been introduced into a suitable cell, the nucleic acid inducesthe synthesis of one or more specified gene products encoded by thenucleic acid. The gene product(s) is(are) other biologicalmacromolecules, such as proteins, not chemically related to the TU. Thenucleic acid TU induces the cell to employ its cellular components toproduce the specific gene product or products encoded by the nucleicacid of the TU. Although any nucleic acid may serve as a TU, in apreferred embodiment, the TU is the DNA of a plasmid or similar vector,wherein the plasmid or vector comprises coding sequences of marker genesor other sequence constructions that facilitate use of the TU forexperimentation and biosynthesis.

As used herein, a “control sequence” is a regulatory nucleotide sequenceincorporated within a TU which interacts with appropriate cellularcomponents of the cell and leads to enhanced or activated biosynthesisof the gene products encoded by the TU. Thus a suitable control sequenceis one with which the components of the cell have the capability tointeract, resulting in synthesis of the gene product. When operablydisposed in a nucleic acid with respect to a specified coding sequence,a control sequence effectively controls expression of the specifiednucleic acid to produce the gene product.

As used herein, a “promoter” is a nucleotide sequence in a TU whichserves as a control sequence.

As used herein, a “Kozak sequence” or “Kozak consensus sequence” is anucleotide sequence at the translational start site which optimizestranslation of eukaryotic mRNAs (Kozak, Mol. Cell. Biology 9: 5134-5142(1989)).

As used herein, a “terminator” is an extended nucleotide sequence whichacts to induce polyadenylation at the 3′ end of a mature mRNA. Aterminator sequence is found after, or downstream from, a particularcoding sequence.

As used herein, a “cell” is a prokaryotic or eukaryotic cell comprisinga TU coding for one or more gene products, or into which such a TU hasbeen introduced. Thus, a cell harbors a foreign or heterologoussubstance, the TU, which is not naturally or endogenously found in thecell as a component. A suitable cell is one which has the capability forthe biosynthesis of the gene products as a consequence of theintroduction of the TU. In particular, a suitable cell is one whichresponds to a control sequence and to a terminator sequence, if any,that may be included within the TU. In important embodiments of thepresent invention, the cell is a mammalian cell. In particularlyimportant embodiments of this invention, the cell is a naturallyoccurring cell in the body of a human or nonhuman subject to whom(which) the TU has been administered as a component of a vaccine.Alternatively, in analytical, or diagnostic applications, includingpreparation of antigen for use as a vaccine or in immunodiagnosticassays, or for demonstrative purposes, the cell may be a human ornonhuman cell cultured in vitro.

As used herein, a “vaccine” or a “composition for vaccinating a subject”specific for a particular pathogen means a preparation, which, whenadministered to a subject, leads to an immunogenic response in asubject. As used herein, an “immunogenic” response is one that confersupon the subject protective immunity against the pathogen. Withoutwishing to be bound by theory, it is believed that an immunogenicresponse may arise from the generation of neutralizing antibodies (i.e.,a humoral immune response) or from cytotoxic cells of the immune system(i.e., a cellular immune response) or both. As used herein, an“immunogenic antigen” is an antigen which induces an immunogenicresponse when it is introduced into a subject, or when it is synthesizedwithin the cells of a host or a subject. As used herein, an “effectiveamount” of a vaccine or vaccinating composition is an amount which, whenadministered to a subject, is sufficient to confer protective immunityupon the subject. Historically, a vaccine has been understood to containas an active principle one or more specific molecular components orstructures which comprise the pathogen, especially its surface. Suchstructures may include surface components such as proteins, complexcarbohydrates, and/or complex lipids which commonly are found inpathogenic organisms.

As used herein, however, it is to be stressed that the terms “vaccine”or “composition for vaccinating a subject” extend the conventionalmeaning summarized in the preceding paragraph. As used herein, theseterms also relate to the TU of the instant invention or to compositionscontaining the TU. The TU induces the biosynthesis of one or morespecified gene products encoded by the TU within the cells of thesubject, wherein the gene products are specified antigens of a pathogen.The biosynthetic antigens then serve as an immunogen. As already noted,the TU, and hence the vaccine, may be any nucleic acid that encodes thespecified immunogenic antigens. In a preferred embodiment of thisinvention, the TU of the vaccine is DNA. The TU can include a plasmid orvector incorporating additional genes or particular sequences for theconvenience of the skilled worker in the fields of molecular biology,cell biology and viral immunology (See Molecular Cloning: A LaboratoryManual, 2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989; and Current Protocols inMolecular Biology, Ausubel et al., John Wiley and Sons, New York 1987(updated quarterly), which are incorporated herein by reference).

The TU molecules of the instant invention comprise nucleic acids, orderivatives of nucleic acids, having nucleotide sequences that encodespecific gene products related to antigens of flaviviruses such as, butnot limited to, WNV, JEV, dengue virus, yellow fever virus and SLEV.Although any nucleic acid may serve as a TU, in an important embodiment,the TU is DNA. Alternatively, the nucleic acids may be RNA molecules.They may also be any one of several derivatives of DNA or RNA having abackbone of phosphodiester bonds that have been chemically modified toincrease the stability of the TU as a pharmaceutical agent.Modifications so envisioned include, but are not limited to,phosphorothioate derivatives or phosphonate derivatives. These and otherexamples of derivatives are well known to persons skilled in the fieldof nucleic acid chemistry.

The genome of JEV has been characterized and sequenced (FIGS. 1 and 2).The M structural protein is expressed as a portion of the polyproteinwhich includes a pre-M sequence (pr). This pr sequence, immediatelyamino terminal to the M protein sequence, prevents conformationalproblems in the processing of the polyprotein. In particular, thepresence of the pr sequence is important in preventing misfolding of theE protein. Thus, the presence of prM allows for assembly of JEVparticles. Once the virion or particle is formed, the pr sequence can becleaved from the prM protein to yield mature virus particles containingM proteins, although cleavage of the prM protein to yield M protein isnot necessary to produce infectious particles. The prM sequences frommany different, related flaviviruses are cleaved to but a low extent,but the flaviviruses themselves are nonetheless, infectious. Examples ofsuch related flaviviruses with similar genomic structures and functionsinclude, but are not limited to WNV, YFV, dengue virus and SLEV.

In one embodiment, the TU encoding flaviviral M and E proteins in theinstant invention is DNA. In accord with the discussion in the precedingparagraph, this DNA comprises a nucleotide sequence which encodes the Mprotein, comprising the pre-M sequence, and a nucleotide sequenceencoding the E protein. In this way, the intended gene products areenabled to form subviral particles within the cell. The pre-M sequencecan then be cleaved in a fashion analogous to that which occurs withrespect to replete virions.

In order to function effectively in vivo as a vaccine, it isadvantageous to include within the TU a control sequence that has theeffect of enhancing or promoting the transcription of the nucleotidesequences encoding the antigens. Use of such promoters is well known tothose of skill in the fields of molecular biology, cell biology andviral immunology (See Molecular Cloning: A Laboratory Manual, 2nd Ed.,Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology,Ausubel et al., John Wiley and Sons, New York 1987 (updated quarterly)).When the TU is used as a vaccine in a mammalian host, the promoter to beemployed is preferably one which operates effectively in mammaliancells. Such a promoter is disposed with respect to the coding sequencesfrom which transcription is to be promoted, at a position at which itmay operably promote such transcription. In a significant embodiment ofthe instant invention, this promoter is the cytomegalovirus earlypromoter. In addition, in a further preferred embodiment of theinvention, the coding sequences are followed, in the TU nucleic acid, bya terminator sequence (Sambrook et al.). Particular embodiments of theinvention relate to both prokaryotic and eukaryotic cells. Many promotersequences are known that are useful in either prokaryotic or eukaryoticcells. (See Sambrook et al.)

The nucleic acids of the invention may further include DNA sequencesknown to those of skill in the art to act as immunostimulatory elements.Examples of such elements include, but are not limited to, certain CpGmotifs in bacterial DNA (Sato et al., Science 273: 352-354 (1996);Klinman et al., Vaccine 17: 19-25 (1998)).

Preparation of the TU of the invention is readily accomplished bymethods well known to workers of skill in the field of molecularbiology. Procedures involved are set forth, for example, in MolecularCloning: A Laboratory Manual, 2nd Ed., Sambrook, Fritsch and Maniatis,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 andCurrent Protocols in Molecular Biology, Ausubel et al., John Wiley andSons, New York 1987 (updated quarterly). The flaviviral RNA molecule maybe isolated from a sample of live virus by methods widely known amongvirologists familiar with flaviviruses, for example, and with othergroups of viruses as well. Methods used with JEV are summarized in Kunoet al. (J. Virol. 72: 73-83 (1998)). The RNA is used as a template forthe synthesis of cDNA using reverse transcriptase. From the cDNA, afragment containing the pre-M through E coding region (FIG. 2) isobtained by digestion with restriction nucleases known to cleave thecDNA appropriately to provide such fragments. Examples of restrictiondigestion of JEV are provided in Nitayaphan et al. (1990) and Konishi etal. (1991). Incorporation of promoters, such as the cytomegaloviruspromoter, sequences to promote efficient translation, such as the Kozaksequence, and of the polyadenylation signal, is likewise well known toskilled practitioners in molecular biology and recombinant DNAengineering (Kozak, Mol. Cell. Biology 9: 5134-5142 (1989); Azevedo etal., Braz. J. Med. Biol. Res. 32: 147-153 (1999)). When a nucleic acidcomprising a TU containing the desired coding sequences and controlsequences is prepared, it may be obtained in larger quantities bymethods that amplify nucleic acids. Such methods are widely known toworkers skilled in molecular biology and recombinant DNA engineering.Examples of these methods include incorporation of the nucleic acid intoa plasmid for replication by culturing in a cell such as a prokaryoticcell and harvesting the plasmid after completing the culture, as well asamplification of the nucleic acid by methods such as PCR and otheramplification protocols, as are well known in the art. These examplesare not intended to limit the ways in which the nucleic acid containingthe TU may be obtained.

The TU-containing nucleic acid molecules of the instant invention may beintroduced into appropriate cells in many ways well known to skilledworkers in the fields of molecular biology and viral immunology. By wayof example, these include, but are not limited to, incorporation into aplasmid or similar nucleic acid vector which is taken up by the cells,or encapsulation within vesicular lipid structures such as liposomes,especially liposomes comprising cationic lipids, or adsorption toparticles that are incorporated into the cell by endocytosis.

In general, a cell of this invention is a prokaryotic or eukaryotic cellcomprising a TU, or into which a TU has been introduced. The TU of thepresent invention induces the intracellular biosynthesis of the encodedprM/M and E antigens. A suitable cell is one which has the capabilityfor the biosynthesis of the gene products as a consequence of theintroduction of the nucleic acid. In particular embodiments of theinvention, a suitable cell is one which responds to a control sequenceand to a terminator sequence, if any, which may be included within theTU. In order to respond in this fashion, such a cell contains within itcomponents which interact with a control sequence and with a terminatorand act to carry out the respective promoting and terminating functions.When the cell is cultured in vitro, it may be a prokaryote, asingle-cell eukaryote or a multicellular eukaryote cell. In particularembodiments of the present invention, the cell is a mammalian cell. Inthese cases, the synthesized prM/M and E protein gene products areavailable for use in analytical, or diagnostic applications, includingpreparation of antigen for use as a vaccine or in immunodiagnosticassays, or for demonstrative purposes.

In some circumstances, such as when the cell is a cultured mammaliancell, the prM/M and E antigens are secreted in the form of subviralparticles. These are aggregates of prM/M and E proteins resembling livevirus in surface ultrastructural morphology and immunogenic properties.Since the TU of the invention does not include the remainder of theflaviviral genome, however, there is no capsid incorporated, and mostimportantly, no infectious viral RNA.

In another important embodiment of this invention, the cell is a naturalcellular component of the subject to whom the TU has been administeredas a vaccine. The TU, when administered to the subject, is taken up bythe cells of the subject. The subject's cells have the capability ofresponding to any promoter sequences, and terminator, if present. In anycase, the TU induces the subject's cells to synthesize flaviviral prM/Mand E gene products. Without wishing to be constrained by theoreticalconsiderations, it is believed that the subject's cells produce subviralparticles in vivo consisting of the prM/M and E antigens, just as hasbeen found to occur with cultured mammalian cells in vitro. Suchsubviral particles, it is believed, then serve as the in vivo immunogen,stimulating the immune system of the subject to generate immunologicalresponses which confer protective immunity on the subject. Again withoutwishing to be limited by theory, the resulting protective immunity mayarise via either humoral or cellular immunity, i.e., via either an MHCclass II- or class I-restricted mechanism, respectively, or by bothmechanisms.

According to the invention, subjects are immunized against infection byflaviviruses, such as JEV, YFV, dengue virus, SLEV, WNV or otherflaviviruses by administering to them an effective amount of a TUcomprising nucleic acid which encodes the prM and/or E antigens. Thenucleic acid, after being incorporated into the cells of the subject,leads to the synthesis of the flaviviral prM/M and/or E antigens.

In order to administer the TU to the subject, it is incorporated into acomposition which comprises a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable” means a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an subject along with the immunogenic material (i.e.,recombinant flavivirus protein antigens or portions thereof) withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of the vaccine inwhich it is contained. Examples of pharmaceutically acceptable carriers,or components thereof, include water, physiological saline and commonphysiological buffers (for further examples, see Arnon, R. (Ed.)Synthetic Vaccines I: pp. 83-92, CRC Press, Inc., Boca Raton, Fla.,1987).

It is understood by those skilled in the art that the critical value indescribing a vaccination dose is the total amount of immunogen needed toelicit a protective response in a host which is subject to infectiousdisease caused by virulent or wild-type flavivirus infection. The numberand volume of doses used can be varied and are determined by thepractitioner based on such parameters as, age, weight, gender, species,type of vaccine to be administered, mode of administration, overallcondition of the subject, et cetera, as well as other important factorsrecognized by those of skill in the art.

The TU may be administered to a subject orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, intranasally, topically orthe like. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of the TU requiredwill vary from subject to subject, depending on the species, age, weightand general condition of the subject, the immunogenicity of the vaccineused, the strain or species of flavivirus against which the subject isbeing immunized, the mode of administration and the like. Thus, it isnot possible to specify an exact amount for every embodiment of thepresent invention. However, an appropriate amount can be determined byone of ordinary skill in the art using only routine experimentationgiven the teachings herein and what is available in the art.

Parenteral administration of the vaccine of the present invention, ifused, is generally characterized by injection. Injectables can beprepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by referenceherein.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc. an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see Remington'sPharmaceutical Sciences (Martin, E. W. (ed.), latest edition, MackPublishing Co., Easton, Pa.).

In one embodiment, the TU of this invention can be administered to thesubject by the use of electrotransfer mediated in vivo gene delivery,wherein immediately following administration of the TU to the subject,transcutaneous electric pulses are applied to the subject, providinggreater efficiency and reproducibility of in vivo nucleic acid transferto tissue in the subject (Mir et al., Proc. Nat. Acad. Sci USA 96:4262-4267 (1999)).

In the methods of the present invention which describe the immunizationof a subject by administering a vaccine of this invention to a subject,the efficacy of the immunization can be monitored according the clinicalprotocols well known in the art for monitoring the immune status of asubject.

An effective amount of a vaccinating composition is readily determinedby those of skill in the art to be an amount which, when administered toa subject, confers protective immunity upon the subject. In order toundertake such a determination, the skilled artisan can assess theability to induce flaviviral prM/M- and E-specific antibodies and/orflaviviral prM/M- and E-specific cytotoxic T lymphocytes present in theblood of a subject to whom the vaccine has been administered. One canalso determine the level of protective immunity conferred upon anexperimental subject by challenge with live flavivirus corresponding tothe antigenic composition used to immunize the experimental subject.Such challenge experiments are well known to those of skill in the art.

In general, in order to immunize a subject against infection by WNV,JEV, YFV, dengue virus, SLEV, or other flaviviruses according to thepresent invention, and recognizing that the TUs employed in such methodsmay have differing overall sizes, doses ranging from about 0.1 μg/kgbody weight to about 50 μg/kg body weight can be used.

It has unexpectedly been found that a TU of the present invention whichis a DNA confers protective immunity at a level of effectivenessapproximating 100% after administration of only a single effective doseof the TU by i.m. injection or by electrotransfer. This is in contrastto many immunization methods carried out using conventional vaccines (asdescribed above), which require one or more booster vaccinations andwhich may not confer protective immunity to an effectiveness near 100%.

It has further been found unexpectedly that protective immunity may betransmitted from a vaccinated female subject to the offspring of thesubject. A significant proportion of neonatal mice was shown to beprotected against viral challenge after the mothers were vaccinatedusing the TU DNA of the invention. Without wishing to be limited bytheory, it is known that passive immunity may be conferred on neonatalmammals due to the presence in maternal milk of neutralizing antibodiesspecific for various pathogens. It is possible that the protectiveimmunity against JEV found within the neonates was transmitted to themin this way.

In another embodiment of the invention, the TU encodes a signal sequenceof a structural protein of a first flavivirus and an immunogenicflavivirus antigen of a second flavivirus. Thus, in one embodiment, forexample, the signal sequence of structural protein of a first flavivirusis replaced by a signal sequence of structural protein of a secondflavivirus, which results in proper folding of the nascent polypeptide,proper processing in a host, and/or proper folding of the processedprotein. In another embodiment of the invention, the TU may encode animmunogenic flavivirus antigen wherein the antigen comprises sequencefrom one or more than one flavivirus.

The present invention further provides immunogenic compositionscomprising the polypeptides of this invention in a pharmaceuticalacceptable carrier for use as a protein vaccine. Antigens produced fromthe transcriptional units of the present invention can be used to eliciteffective immune responses in a subject. Antigens for this purpose cancomprise flavivirus prM protein, flavivirus M protein, flavivirus Eprotein or any combination thereof, including immunogenic fragments ofthe proteins. A particularly preferred embodiment is the use of the NRAdescribed herein. A further preferred embodiment is a chimeric proteincomprising the signal sequence of one flavivirus and the structuralprotein(s) of one or more different flaviviruses. In a particularlypreferred embodiment, the signal sequence of the antigen is the Japaneseencephalitis virus signal sequence.

In other embodiments, the protein vaccine of this invention furthercomprises a suitable adjuvant. As used herein, an “adjuvant” is apotentiator or enhancer of the immune response. The term “suitable ” ismeant to include any substance which can be used in combination with thevaccine immunogen (i.e., flavivirus prM protein, flavivirus M protein,flavivirus E protein, or any combination thereof) to augment the immuneresponse, without producing adverse reactions in the vaccinated subject.Effective amounts of a specific adjuvant may be readily determined so asto optimize the potentiation effect of the adjuvant on the immuneresponse of a vaccinated subject. In a preferred embodiment, adjuvantingof the vaccines of this invention is a 2-stage process, utilizing firsta 2% aluminum hydroxide solution and then a mineral oil. In specificembodiments, suitable adjuvants can be chosen from the following group:mineral, vegetable or fish oil with water emulsions, incomplete Freund'sadjuvant, E. coli J5, dextran sulfate, iron oxide, sodium alginate,Bacto-Adjuvant, certain synthetic polymers such as Carbopol (BF GoodrichCompany, Cleveland, Ohio), poly-amino acids and co-polymers of aminoacids, saponin, carrageenan, REGRESSIN (Vetrepharm, Athens, Ga.),AVRIDINE (N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine),long chain polydispersed β(1,4) linked mannan polymers interspersed withO-acetylated groups (e.g. ACEMANNAN), deproteinized highly purified cellwall extracts derived from non-pathogenic strain of Mycobacteriumspecies (e.g. EQUIMUNE, Vetrepharm Research Inc., Athens Ga.), Mannitemonooleate, paraffin oil and muramyl dipeptide.

In another aspect, this invention provides a method for immunizingsubjects with immunogenic amounts of the protein vaccine of theinvention to elicit an effective immune response in the subject.Immunization can be carried out orally, parenterally, intranasally,intratracheally, intramuscularly, intramammarily, subcutaneously,intravenously and/or intradermally. The vaccine containing theflavivirus prM protein, flavivirus M protein and/or the flavivirus Eprotein can be administered by injection, by inhalation, by ingestion,or by infusion. A single dose can be given and/or repeated doses of thevaccine preparations, i.e. “boosters,” can be administered at periodictime intervals to enhance the initial immune response or after a longperiod of time since the last dose. The time interval betweenvaccinations can vary, depending on the age and condition of thesubject.

The term “immunogenic amount” means an amount of an immunogen, or aportion thereof, which is sufficient to induce an immune response in avaccinated subject and which protects the subject against disease causedby wild-type or virulent flavivirus infections upon exposure thereto orwhich has a therapeutic or commercially beneficial effect that lessensthe effect of flavivirus infection on the vaccinated subject.

The invention further provides an antibody produced in response toimmunization by the antigen of this invention. The antibodies of thepresent invention can include polyclonal and monoclonal antibodies whichcan be intact immunoglobulin molecules, chimeric immunoglobulinmolecules, “humanized antibodies,” or Fab or F(ab′)₂ fragments. Suchantibodies and antibody fragments can be produced by techniques wellknown in the art which include those described in Harlow and Lane(Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1989) and Kohler et al. (Nature 256:495-97, 1975)and U.S. Pat. Nos. 5,545,806, 5,569,825 and 5,625,126, incorporatedherein by reference. The antibodies can be of any isotype IgG, IgA, IgD,IgE and IgM.

The present invention can also include single chain antibodies (ScFv),comprising linked V_(H) and V_(L) domains and which retain theconformation and specific binding activity of the native idiotype of theantibody. Such single chain antibodies are well known in the art and canbe produced by standard methods. (see, e.g., Alvarez et al., Hum. GeneTher. 8: 229-242 (1997)).

Antibodies can be produced against the antigens of this invention whichare synthesized from nucleic acid sequences encoding immunogenic aminoacid sequences of the prM, M and/or E antigens of one or moreflaviviruses and the signal sequence of a different flavivirus (e.g.,JEV). Immunogenic peptides synthesized from the use of these chimericconstructs can easily be identified by use of methods well known in theart for identifying immunogenic regions in an amino acid sequence andused to produce the antibodies of this invention.

Conditions whereby an antigen/antibody complex can form, as well asassays for the detection of the formation of an antigen/antibody complexand quantitation of the detected protein, are standard in the art. Suchassays can include, but are not limited to, Western blotting,immunoprecipitation, immunofluorescence, immunocytochemistry,immunohistochemistry, fluorescence activated cell sorting (FACS),fluorescence in situ hybridization (FISH), immunomagnetic assays, ELISA,ELISPOT (Coligan et al., eds. 1995. Current Protocols in Immunology.Wiley, New York.), agglutination assays, flocculation assays, cellpanning, etc., as are well known to the artisan.

As used herein, the term “bind” means the well characterized binding ofantibody to antigen as well as other nonrandom association with anantigen. “Specifically bind” as used herein describes an antibody orother ligand that does not cross react substantially with any antigenother than the one specified, which in this case, is an antigen of thisinvention.

The antibody or ligand of this invention can be bound to a substrate(e.g., beads, tubes, slides, plates, nitrocellulose sheets, etc.) orconjugated with a detectable moiety or both bound and conjugated. Thedetectable moieties contemplated for the present invention can include,but are not limited to, an immunofluorescent moiety (e.g., fluorescein,rhodamine), a radioactive moiety (e.g., ³²P, ¹²⁵I, ³⁵S), an enzymemoiety (e.g., horseradish peroxidase, alkaline phosphatase), a colloidalgold moiety and a biotin moiety. Such conjugation techniques arestandard in the art (for example, Harlow and Lane, Antibodies: ALaboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989); Yang et al., Nature 382: 319-324 (1996)).

The present invention further provides a method of detecting flavivirusantibody in a sample, comprising contacting the sample with theflavivirus antigen of the present invention, under conditions whereby anantigen/antibody complex can form; and detecting formation of thecomplex, thereby detecting flavivirus antibody in the sample.

The present invention further provides a method of detecting flavivirusantigen in a sample, comprising contacting the sample with an antibodyof this invention under conditions whereby an antigen/antibody complexcan form; and detecting formation of the complex, thereby detectingflavivirus antigen in the sample.

The method of detecting flavivirus antigen in a sample can be performed,for example, by contacting a fluid or tissue sample from a subject withan antibody of this invention and detecting binding of the antibody tothe antigen. It is contemplated that the antigen will be on an intactflavivirus virion, will be a flavivirus-encoded protein displayed on thesurface of a flavivirus-infected cell expressing the antigen, or will bea fragment of the antigen. A fluid sample of this method can compriseany biological fluid which could contain the antigen or a cellcontaining the antigen, such as cerebrospinal fluid, blood, bile,plasma, serum, saliva and urine. Other possible examples of body fluidsinclude sputum, mucus and the like.

The method of detecting flavivirus antibody in a sample can beperformed, for example, by contacting a fluid or tissue sample from asubject with an antigen of this invention and detecting the binding ofthe antigen to the antibody. A fluid sample of this method can compriseany biological fluid which could contain the antibody, such ascerebrospinal fluid, blood, bile, plasma, serum, saliva and urine. Otherpossible examples of body fluids include sputum, mucus and the like.

Enzyme immunoassays such as immunofluorescence assays (IFA), enzymelinked immunosorbent assays (ELISA) and immunoblotting can be readilyadapted to accomplish the detection of flavivirus antibodies accordingto the methods of this invention. An ELISA method effective for thedetection of the antibodies can, for example, be as follows: (1) bindthe antigen to a substrate; (2) contact the bound antigen with a fluidor tissue sample containing the antibody; (3) contact the above with asecondary antibody bound to a detectable moiety which is reactive withthe bound antibody (e.g., horseradish peroxidase enzyme or alkalinephosphatase enzyme); (4) contact the above with the substrate for theenzyme; (5) contact the above with a color reagent; and (6)observe/measure color change or development.

Another immunologic technique that can be useful in the detection offlavivirus antibodies uses monoclonal antibodies (MAbs) for detection ofantibodies specifically reactive with flavivirus antigens in acompetitive inhibition assay. Briefly, sample is contacted with anantigen of this invention which is bound to a substrate (e.g., an ELISA96-well plate). Excess sample is thoroughly washed away. A labeled(e.g., enzyme-linked, fluorescent, radioactive, etc.) monoclonalantibody is then contacted with any previously formed antigen-antibodycomplexes and the amount of monoclonal antibody binding is measured. Theamount of inhibition of monoclonal antibody binding is measured relativeto a control (no antibody), allowing for detection and measurement ofantibody in the sample. The degree of monoclonal antibody inhibition canbe a very specific assay for detecting a particular flavivirus varietyor strain, when based on monoclonal antibody binding specificity for aparticular variety or strain of flavivirus. MAbs can also be used fordirect detection of flavivirus antigens in cells by, for example,immunofluorescence assay (IFA) according to standard methods.

As a further example, a micro-agglutination test can be used to detectthe presence of flavivirus antibodies in a sample. Briefly, latex beads,red blood cells or other agglutinable particles are coated with theantigen of this invention and mixed with a sample, such that antibodiesin the sample that are specifically reactive with the antigen crosslinkwith the antigen, causing agglutination. The agglutinatedantigen-antibody complexes form a precipitate, visible with the nakedeye or measurable by spectrophotometer. In a modification of the abovetest, antibodies of this invention can be bound to the agglutinableparticles and antigen in the sample thereby detected.

The present invention further provides a method of diagnosing aflavivirus infection in a subject, comprising contacting a sample fromthe subject with the antigen of this invention under conditions wherebyan antigen/antibody complex can form; and detecting antigen/antibodycomplex formation, thereby diagnosing a flavivirus infection in asubject.

The present invention further provides a method of diagnosing aflavivirus infection in a subject, comprising contacting a sample fromthe subject with the antibody of this invention under conditions wherebyan antigen/antibody complex can form; and detecting antigen/antibodycomplex formation, thereby diagnosing a flavivirus infection in asubject.

In the diagnostic methods taught herein, the antigen of this inventioncan be bound to a substrate and contacted with a fluid sample such asblood, serum, urine or saliva. This sample can be taken directly fromthe patient or in a partially purified form. In this manner, antibodiesspecific for the antigen (the primary antibody) will specifically reactwith the bound antigen. Thereafter, a secondary antibody bound to, orlabeled with, a detectable moiety can be added to enhance the detectionof the primary antibody. Generally, the secondary antibody or otherligand, which is reactive, either specifically with a different epitopeof the antigen or nonspecifically with the ligand or reacted antibody,will be selected for its ability to react with multiple sites on theprimary antibody. Thus, for example, several molecules of the secondaryantibody can react with each primary antibody, making the primaryantibody more detectable.

The detectable moiety allows for visual detection of a precipitate or acolor change, visual detection by microscopy, or automated detection byspectrometry, radiometric measurement or the like. Examples ofdetectable moieties include fluorescein and rhodamine (for fluorescencemicroscopy), horseradish peroxidase (for either light or electronmicroscopy and biochemical detection), biotin-streptavidin (for light orelectron microscopy) and alkaline phosphatase (for biochemical detectionby color change).

Particular embodiments of the present invention are set forth in theexamples which follow. These examples are not intended to limit thescope of the invention as disclosed in this specification.

EXAMPLES

General methods utilizing molecular biology and recombinant DNAtechniques related to preparing and expressing the nucleic acid TUmolecules of the invention are set forth in, for example, CurrentProtocols in Molecular Biology, Ausubel et al., John Wiley and Sons, NewYork 1987 (updated quarterly), and Molecular Cloning: A LaboratoryManual 2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989.

Example 1

Preparation of recombinant plasmids containing the transcriptional unitencoding JEV prM and E antigens. Genomic RNA was extracted from 150 μLof JEV strain SA 14 virus seed grown from mouse brain using a QIAamp™Viral RNA Kit (Qiagen, Santa Clarita, Calif.). RNA, adsorbed on a silicamembrane, was eluted in 80 μL of nuclease-free water, and used as atemplate for the amplification of JEV prM and E gene coding sequences.Primer sequences were obtained from the work of Nitayaphan et al.(Virology 177: 541-552 (1990)). A single cDNA fragment containing thegenomic nucleotide region 389-2478 was amplified by the reversetranscriptase-polymerase chain reaction (RT-PCR). Restriction sites KpnIand XbaI, the consensus Kozak ribosomal binding sequence, and thetranslation initiation site were engineered at the 5′ terminus of thecDNA by amplimer 14DV389 (nucleotide sequence, SEQ ID NO:1; amino acidsequence, SEQ ID NO:2). An in-frame translation termination codon,followed by a NotI restriction site, was introduced at the 3′ terminusof the cDNA by amplimer c14DV2453 (SEQ ID NO:3) (FIG. 2). One-tubeRT-PCR was performed using a Titan RT-PCR Kit (Boehringer Mannheim,Indianapolis, Ind.). 10 μL of viral RNA was mixed with 1 μL each of14DV389 (50 μM) and c14DV2453 (50 μM) and 18 μL of nuclease-free waterand the mixture was heated at 85° C. for 5 min and then cooled to 4° C.75 μL of reaction mix [20 μL 5× buffer, 2 μL of dNTP mixture (10 mMeach), 5 μL of dithiothreitol (0.1 mM), 0.5 μL of RNasin™ (40 U/μL,Boehringer Mannheim), 2 μL of polymerase mixture, and 45.5 μL ofnuclease-free water] was added and RT-PCR performed as follows: 1 cycle(50° C. for 30 min, 94° C. for 3 min, 50° C. for 30 s, 68° C. for 2.5min), 9 cycles (94° C. for 30 s, 50° C. for 30 s, 68° C. for 2.5 min),20 cycles (94° C. for 30 s, 50° C. for 30 s, 68° C. for 2.5 min in thefirst cycle, with an increment of 5 s per cycle thereafter), and a finalextension at 68° C. for 15 min. The RT-PCR product was purified by aQIAquick™ PCR Purification Kit (Qiagen) and eluted with 50 μL of 1 mMTris-HCl, pH 7.5.

All vector constructions and analyses were carried out by using standardtechniques (Sambrook et al., 1989). RT-PCR amplified cDNA, digested withKpnI and NotI nucleases, was inserted into the KpnI-NotI site ofeukaryotic expression plasmid vector (pCDNA3, Invitrogen, Carlsbad,Calif.). Electroporation-competent Escherichia coli XL1-Blue cells(Stratagene, La Jolla, Calif.) were transformed by electroporation (GenePulser™, Bio-Rad, Hercules, Calif.) and plated onto LB agar platescontaining 100 μg/mL carbenicillin (Sigma Chemical Co., St. Louis, Mo.).Clones were picked and inoculated into 3 mL LB broth containing 100μg/mL carbenicillin. Plasmid DNA was extracted from a 14 h culture usinga QIAprep™ Spin Miniprep Kit (Qiagen). Automated DNA sequencing wasperformed as recommended (Applied Biosystems/Perkin Elmer, Foster City,Calif.). Both strands of the cDNA were sequenced and shown to beidentical to the sequence for the original SA14 strain (Nitayaphan etal., 1990).

The fragment of plasmid pCDNA3 (Invitrogen, Carlsbad, Calif.) fromnucleotide (nt) 1289 to nt 3455, containing f1 ori, SV40 ori, theneomycin resistance gene, and SV40 poly(A) elements was deleted by PvuIIdigestion and then ligated to generate the pCBamp plasmid. The vectorpCIBamp, containing a chimeric intron insertion at the NcoI/KpnI site ofthe pCBamp was constructed by excising the intron sequence from pCI(Promega, Madison, Wis.) by digestion with NcoI and KpnI. The resulting566-bp fragment was cloned into pCBamp by digesting with NcoI-KpnI toreplace its 289-bp fragment. FIG. 3 presents the relationships betweenthe plasmids pCDA3, pCBamp, and pCIBamp.

Plasmids containing the transcriptional unit encoding JEV prM and Eproteins were prepared from these plasmids. The cDNA fragment containingthe JEV prM and E coding regions in the recombinant plasmid pCDJE2-7(nucleotide sequence, SEQ ID NO:10; amino acid sequence, SEQ ID NO:11),derived from the pCDNA3 vector, was excised by digestion with NotI andKpnI or XbaI and cloned into the KpnI-NotI site of pCBamp, pCIBamp,pCEP4 (Invitrogen, Carlsbad, Calif.), or pREP4 (Invitrogen, Carlsbad,Calif.), or into the SpeI-NotI site of pRc/RSV (Invitrogen, Carlsbad,Calif.) expression vector to create pCBJE1-14 (nucleotide sequence, SEQID NO:17; amino acid sequence, SEQ ID NO:18), pCIBJES14, pCEJE, pREFE,and pRCJE, respectively. Both strands of the cDNA from clones of eachplasmid were sequenced and recombinant clones with the correctnucleotide sequence were identified. Plasmid DNA for use in the in vitrotransformation of mammalian cells or mouse immunization experiments waspurified by anion exchange chromatography using an EndoFree™ PlasmidMaxi Kit (Qiagen).

Example 2

Evaluation of JEV prM and E proteins expressed by various recombinantplasmids using an indirect immunofluorescent antibody assay. Theexpression of JEV specific gene products by the various recombinantexpression plasmids was evaluated in transiently transfected cell linesof COS-1, COS-7 and SV-T2 (ATCC, Rockville Md.; 1650-CRL, 1651-CRL, and163.1-CCL, respectively) by indirect immunofluorescent antibody assay(IFA). The SV-T2 cell line was excluded from further testing since apreliminary result showed only 1-2% of transformed SV-T2 cells were JEVantigen positive. For transformation, cells were grown to 75% confluencein 150 cm² culture flasks, trypsinized, and resuspended at 4° C. inphosphate buffered saline (PBS) to a final cell count 5×10⁶ per mL. 10μg of plasmid DNA was electroporated into 300 μL of cell suspensionusing a BioRad Gene Pulse™ (Bio-Rad) set at 150 V, 960 μF and 100 Ωresistance. Five minutes after electroporation, cells were diluted with25 mL fresh medium and seeded into a 75 cm² flask. 48 h aftertransformation the medium was removed from the cells, and the cells weretrypsinized and resuspended in 5 mL PBS with 3% normal goat serum. 10 μLaliquots were spotted onto slides, air dried and fixed with acetone at−20° C. for 20 min. IFA was performed with acetone-fixedplasmid-transformed cells using fluorescein isothiocyanate-conjugatedgoat anti-mouse immunoglobulin G (Sigma Chemical Co.) and JEV HIAF.

To determine the influence of various promoter and poly(A) elements onthe JEV prM and E protein expression, COS-1 and COS-7 cell lines weretransiently transformed by an equal amount of pCDJE2-7 (SEQ ID NO:10),pCEJE, pREJE, or pRCJE plasmid DNA. JEV antigens were expressed in bothcell lines transformed by all four recombinant plasmids, thus confirmingthat the CMV or RSV (rous sarcoma virus) promoter and BGH or SV40poly(A) elements were functionally active. However, the percentage oftransformed cells and the level of JEV antigens expressed, as determinedby the number of IFA positive cells and IFA intensity, respectively,differed greatly among the various plasmids (Table 1). A significantlyhigh percentage of COS-1 cells transformed by pCDJE2-7 (SEQ ID NO:10),pCBJE1-14 (SEQ ID NO:17) and pCIBJES14 expressed the JEV antigens, andthe level of the expressed proteins was compatible with JEV-infectedcells. Cells transfected with pCEJE, pREJE, or pRCJE vectors, on theother hand, had a low percentage of antigen-expressing cells, as well asa low intensity of fluorescence, indicating weak expression of theantigens.

In order to ascertain whether the enhanced expression of JEV proteins bypCDJE2-7 (SEQ ID NO:10) was influenced by the SV40-encoded eukaryoticorigin of replication, the plasmid pCBJE1-14 (SEQ ID NO:17) wasconstructed so that a 2166-bp fragment, containing f1 ori, SV40 ori, theneomycin resistance gene and SV40 poly(a) elements from pCDJE2-7, wasdeleted. A chimeric intron was then inserted into pCBJE1-14 to generatepCIBJES14. The pCIBJES14 plasmid was used to determine if the expressionof JEV proteins could be enhanced by the intron sequence. Followingtransformation, cells harboring both pCBJE1-14 and pCIBJES14 vectorsexpressed a level of JEV antigens similar to that observed with pCDJE2-7(Table 1). This result indicates that expression of JEV prM and Eantigens by recombinant vectors is influenced only by thetranscriptional regulatory elements. Neither the eukaryotic origin ofreplication nor the intron sequence enhanced JEV antigen expression inthe cells used. Vectors containing the CMV promoter and BGH poly(A)(FIG. 3) were selected for further analysis.

Example 3

Selection of an in vitro transformed, stable cell line constitutivelyexpressing JEV specific gene products. COS-1 cells were transformed with10 μg of pCDJE2-7 DNA by electroporation as described in the previousexample. After a 24 hr incubation in non-selective culture medium, cellswere treated with neomycin (0.5 mg/mL, Sigma Chemical Co.).Neomycin-resistant colonies, which became visible after 2-3 weeks, werecloned by limited dilution in neomycin-containing medium. Expression ofvector-encoded JEV gene products was initially screened by IFA using JEVHIAF. One JEV-IFA positive clone (JE-4B) and one negative clone (JE-5A)were selected for further analysis and maintained in medium containing200 μg/mL neomycin.

Authenticity of the JEV E protein expressed by the JE-4B clone wasdemonstrated by epitope mapping by IFA using a panel of JEV E-specificmurine monoclonal antibodies (Mab) (Kimura-Kuroda et al., J. Virol. 45:124-132 (1983); Kimura-Kuroda et al., J. Gen. Virol. 67: 2663-2672(1986); Zhang et al., J. Med. Virol. 29: 133-138 (1989); and Roehrig etal., Virol. 128: 118-126 (1983)). JEV HIAF and normal mouse serum wereused as positive and negative antibody controls, respectively. FourJEV-specific, six flavivirus-subgroup specific, and two flavivirus-groupreactive Mabs reacted similarly with the 4B clone or JEV-infected COS-1cells (Table 2).

Example 4 Antigenic Properties and Immunological Detection of SubviralParticles Secreted by the JE-4B COS-1 Cell Line

a. Preparation of subviral particles. JE-4B COS-1 cells were grown andmaintained in medium containing 200 μg/mL of neomycin. The culturedmedium was routinely harvested and stored at 4° C., and replenishedtwice weekly, and the cells were split 1:5 every 7-10 days. Culturemedium was clarified by centrifugation at 10,000 rpm for 30 min in aSorvall F16/250 rotor at 4° C., and centrifuged further for 4 hr at39,000 rpm in a Sorvall TH641 rotor at 4° C. through a 5% sucrosecushion (w/w, prepared with 10 mM Tris HCl, pH 7.5, 100 mM NaCl (TNbuffer)). The pellet containing subviral particles was resuspended in TNbuffer and stored at 4° C. Alternatively, 7% or 10% PEG-8000 (w/v) wasadded to the clarified culture medium. The mixture was stirred at 4° C.for at least 2 hr, and the precipitated particles were collected bycentrifugation at 10,000 rpm for 30 min. The precipitate was resuspendedin TN buffer and stored at 4° C. The subviral particles were purifiedfrom both pelleted and PEG-precipitated preparations by rate zonalcentrifugation in a 5-25% continuous sucrose gradient in TN at 38,000rpm at 4° C. for 90 min. 1-mL fractions were collected from the top ofthe gradient, tested by antigen capture ELISA (see below), and thepositive fractions loaded onto a 25-50% sucrose gradient in TN. This wascentrifuged overnight in an equilibrium density centrifugation at 35,000rpm at 4° C. 0.9-mL fractions from the equilibrium gradients werecollected from the bottom. They were tested by antigen-capture ELISA andassessed for hemagglutination (HA) activity at pH 6.6. An aliquot of 100μL of each fraction was weighed precisely to determine its density. TheELISA-positive fractions were pooled and pelleted at 39,000 rpm at 4° C.for 3-4 hr and the pellet resuspended in TN buffer. Antigen-captureELISA and HA titers were determined on the pelleted samples.JEV-infected COS-1 cell supernatant was also subjected to similarpurification protocols as detailed above and used as a positive controlfor the gradient analysis. JE virions were also purified from infectedC6/36 cells 5-6 days postinfection by sedimentation in aglycerol/tartrate equilibrium gradient.

b. Western blots of subviral particles. Gradient-purified samples of thesubviral particles were mixed with electrophoresis sample buffer and runon 10 or 12.5% sodium dodecyl sulfate-containing polyacrylamide gels(SDS-PAGE) as described by Laemmli (Nature 277: 680-685 (1970)).Proteins were transferred to a nitrocellulose membrane andimmunochemically detected with polyclonal JEV HIAF, flaviviruscross-reactive anti-E Mab 4G2 (Henchal et al., Amer. J. Trop. Med. Hyg.31: 830-836 (1982)), or mouse anti-prM peptide hyperimmune serum (JM01).FIG. 4 shows a comparison of the M and E proteins produced by JEVinfected C6/36 and JE-4B COS-1 cells. Some nonspecific reactivity to Eprotein was observed in the normal mouse ascitic fluid and Jmolanti-peptide serum. Proteins identical in size to M and E were secretedin the subviral particles and could be detected by E-specific Mab 4G2and prM-specific JM01 antiserum, respectively.

c. Density gradient detection of JEV subviral particles in culturemedium. For ELISA, antigen-capture antibody (4G2) was diluted in 0.1 Msodium carbonate buffer, pH 9.6, and used to coat 96-well microtiterplates (Immulon II, Dynatech. Chantilly, Va.) by overnight incubation at4° C. After blocking with 3% normal goat serum in PBS, two-foldserially-diluted samples were added to the 4G2-coated plate andincubated 1.5 hours at 37° C. Captured antigen was detected byhorseradish peroxidase-conjugated 6B6C-1 Mag, and incubated for 1 hourat 37° C. The enzyme activity on the solid phase was then detected withTMB (3,3′,5,5′-tetramethylbenzidine)-ELISA (Life Technologies, GrandIsland, N.Y.).

Approximately 500 mL of cell culture medium from 15×150 cm² flasks ofJE-4B cells was collected four days after cells were seeded.PEG-precipitated subviral particles were resuspended in 2 mL of TNbuffer, pH 7.5; a 0.7 mL aliquot of this resuspended pellet was loadedonto a 5-25% sucrose gradient. Triton X-100, which disrupts subviralparticles, was added to another 0.7 mL aliquot to a final concentrationof 0.1% and this was loaded onto a 5-25% sucrose gradient prepared in TNbuffer containing 0.1% Triton X-100. A definite opaque band was observedapproximately 2.5 cm from the top of the gradient containing TritonX-100, but not in the gradient without detergent. Fractions (1 mL) werecollected from top to bottom for each gradient (FIG. 5). Each collectedfraction was analyzed by antigen capture ELISA. Antigen was detected infractions 4-6, indicating relatively rapid sedimentation characteristicof subviral particles. Treatment of the PEG precipitate from JE-4Bculture medium with Triton X-100 shifted the position of ELISA-reactivematerial to the top of the gradient. Thus treatment with Triton X-100produces only slow-sedimenting molecules. A similar finding was reportedby Konishi et al. (Virol. 188: 714-720 (1992)). These results show thatrapidly sedimenting subviral particles containing prM/M and E could bedisrupted by detergent treatment.

Hemagglutination (HA) activity was determined in the pH range from 6.1to 7.0 by the method of Clarke and Casals (Amer. J. Trop. Med. Hyg. 7:561-573 (1958)). The subviral particle secreted by JE-4B cells and thevirion particle produced by JEV infected COS-1 cells had a similar HAprofile with the optimum pH determined to be 6.6.

Example 5

Comparison of the immune response in mice vaccinated with pCDJE2-7nucleic acid vaccine of the invention and commercial JEV vaccine. Groupsof five 3-week-old female, ICR outbred mice were injectedintramuscularly in the left and right quadriceps with 100 μg of pCDJE2-7plasmid in 100 μL of dH₂O or were given doses of JE-VAX (manufactured bythe Research Foundation for Microbial Disease of Osaka University anddistributed by Connaught Laboratories, Swiftwater, Pa.) subcutaneouslythat are one-fifth the dose given to humans. The plasmid pCDNA3/CAT(Invitrogen), which encodes and expresses an unrelated protein, was usedas the negative vaccination control. Except for one group ofpCDJE2-7-vaccinated mice, all animals were boosted 3 weeks later with anadditional dose of plasmid or JE-VAX. Mice were bled from theretroorbital sinus at 3, 6, 9, 23, 40 and 60 weeks after inoculation.JEV antibody titers were determined by enzyme-linked imunosorbent assay(ELISA) against purified JEV or by plaque reduction neutralization tests(PRNT) (Roehrig et al., Virol. 171: 49-60 (1989); and Hunt and Calisher,Amer. J. Trop. Med. Hyg. 28: 740-749 (1979)).

The pCDJE2-7 nucleic acid vaccine and JE-VAX provided 100%seroconversion within three weeks after the first vaccination in allthree groups of mice (Table 3). The JEV ELISA and PRNT antibody titersreached the highest level at week 6 and week 9, respectively, afterimmunization. Mice receiving 1 dose of DNA vaccine had similar antibodyresponses as those receiving 2 doses. Comparable ELISA antibody titerswere maintained in DNA-vaccinated groups up to 60 weeks, after which theexperiment was terminated. However, only one of four mice in the JE-VAXgroup was JEV antibody positive at 60 weeks post-inoculation. ThepCDNA3/CAT control group did not have any measurable JEV antibody. Theseresults demonstrate that a single dose of JEV-specific nucleic acidvaccine is more effective in maintaining JEV antibody in mice than thecommercial, FDA-approved JE-VAX vaccine.

Example 6

Comparison of various nucleic acid vaccine constructs of the inventionand commercial JEV vaccine for effectiveness of vaccination at differentages. A similar level of JEV protein was expressed by COS-1 cellstransformed by either pCDJE2-7, pCBJE1-14, or pCIBJES14. JEV antibodyinduction by these nucleic acid constructs was compared to JE-VAXcommercial vaccine at two different ages at vaccination. Three-day(mixed sex) or 3-week-old (female) ICR outbred mice, 10 per group, werevaccinated intramuscularly with 50 or 100 μg of plasmid DNA, orsubcutaneously with doses of JE-VAX that are one-tenth or one-fifth thedose given to humans. Serum specimens were collected at 3 and 7 weeksafter immunization and tested at a 1:1600 dilution by ELISA usingpurified JEV as an antigen. Results are shown in Table 4.

Plasmid pCBJE1-14 provided the highest extent of seroconversion, i.e.,antibody titer greater than 1:1600, achieving 80-100% at both ages ofvaccination. Administration of pCDJE2-7 or pCIBJES14 provided moderateseroconversion by 7 weeks when 3-day old mice were vaccinated (60% foreach), but weaker seroconversion (40% and 10%, respectively) whenmeasured 3 weeks after vaccination. When these plasmids wereadministered at the age of 3 weeks, however, seroconversions of 90% or100% were attained at both 3 weeks and 7 weeks after vaccination. Incontrast, the commercial vaccine, JE-VAX, conferred no seroconversionwhen administered at 3 days of age, and 100% when given at 3 weeks ofage. Thus the nucleic acid TU's for JEV prM and E provided an extent ofseroconversion better than a very high dose of the commercial vaccine,and unexpectedly high seroconversion in both young and more matureanimals.

Example 7

Protective immunity conferred by the nucleic acid vaccine of theinvention. Three-day old vaccinated groups from Example 6 werechallenged 7 weeks after vaccination by intraperitoneal injection of50,000 pfu/100 μL of the mouse-adapted JEV strain SA14 and observed for3 weeks. 100% protection was achieved in groups that received variousnucleic acid TU-containing vaccine constructs for up to 21 days (Table5). In contrast, 60% of the JE-VAX-vaccinated mice, as well as 70% ofthe pCDNA3/CAT-vaccinated negative controls, did not survive viruschallenge by 21 days. These results indicate that the nucleic acid TU'sof the invention confer unexpectedly effective protection on vaccinatedmice. This suggests the possibility of employing the nucleic acidvaccine of the invention as an early childhood vaccine for humans. Incontrast, JE-VAX, the inactivated human vaccine currently used, does notappear to be effective in young animals.

Example 8

Passive protection of neonatal mice correlated with the maternalantibody titer. Female ICR mice at the age of 3 weeks were vaccinatedwith either one dose or two doses spaced two days apart of pCDJE2-7plasmid DNA, at 100 μg/100 μL, or with two doses of JE-VAX that wereone-fifth the dose given to humans. The negative control group receivedtwo doses of 100 μg/100 μL of pCDNA-3/CAT plasmid. Passive protection bymaternal antibody was evaluated in pups resulting from matings ofexperimental females with non-immunized male mice that occurred nineweeks following the first vaccination or 6 weeks following the secondvaccination. Pups were challenged between 3-15 days after birth byintraperitoneal administration of 5,000 pfu/100 μL of mouse-adapted SA14virus and observed daily for 3 weeks (Table 6). The survival ratescorrelated with the maternal neutralizing antibody titers. 100% of pupsnursed by mothers with a PRNT of 1:80 survived viral infection, whereasnone of the pups from the control mother survived (Table 6). Partialprotection of 45% and 75% was observed in older pups that were nursed bymothers with a PRNT titer of 1:20 and 1:40, respectively. The survivalrates also correlated with the length of time that pups were nursed bythe immune mother. As just indicated, 13-15 day old pups had highsurvival rates. None of the 3-4 day old pups, however, survived viruschallenge when the mother had a PRNT titer of 1:20 or 1:40. Thusmaternal antibody provides partial to complete protective immunity tothe offspring. In addition, JEV antibody was detected by ELISA in thesera of 97% (29/30) of the post-challenge pups.

Mice were inoculated intramuscularly with 1 or 2, 100 μg doses ofplasmid DNA, or subcutaneously with two, ⅕ human doses of JE-VAXvaccine. Sera were collected 9 weeks post-vaccination for PRNT testingprior to mating with non-immune male.

Example 9

Preparation of recombinant plasmids containing the transcriptional unitencoding WNV prM and E antigens. Genomic RNA was extracted from 150 μLof Vero cell culture medium infected with NY 99-6480 strain, an strainisolated from the outbreak in New York 1999, using the QIAamp™ Viral RNAKit (Qiagen, Santa Clarita, Calif.). Extracted RNA was eluted andsuspended in 80 μl of nuclease-free water, and used as a template forthe amplification of WNV prM and E gene coding sequences. Primersequences were obtained from the work of Lanciotti et al. (Science 286:2333-2337 (1999)). A cDNA fragment containing the genomic nucleotideregion was amplified by the reverse transcriptase-polymerase chainreaction (RT-PCR). Restriction sites BsmBI and KasI were engineered atthe 5′ terminus of the cDNA by using amplimer WN466 (nucleotidesequence, SEQ ID NO:12). An in-frame translation termination codon,followed by a NotI restriction site was introduced at the 3′ terminus ofthe cDNA by using amplimer cWN2444 (SEQ ID NO:13). The RT-PCR productwas purified by a QIAquick™ PCR Purification Kit (Qiagen).

The double-stranded amplicon produced by use of the two amplimers above(SEQ ID NO:12 and SEQ ID NO:13) was digested with KasI and NotI enzymesto generate a 998 bp (nt-1470 to 2468) fragment of DNA was inserted intothe KasI and NotI sites of a pCBJESS vector to form an intermediateplasmid, pCBINT. The pCBJESS was derived from the pCBamp plasmid, thatcontained the cytomegalovirus early gene promoter and translationalcontrol element and an engineered JE signal sequence element (Chang etal., J. Virol. 74: 4244-4252 (2000)). The JE signal sequence elementcomprises the JE signal sequence (SEQ ID NO:14).

The cDNA amplicon was subsequently digested with BsmBI and Kas I enzymesand the remaining 1003 bp fragment (nt-466 to 1470) was inserted in tothe KasI site of pCBINT to form pCBWN (nucleic acid sequence, SEQ IDNO:15; amino acid sequence, SEQ ID NO:16). Automated DNA sequencingusing an ABI prism 377 Sequencer (Applied Biosystems/Perkin Elmer,Foster City, Calif.) was used to confirm that the recombinant plasmidhad a correct prM and E sequence as defined by Lanciotti et al. (Science286: 2333-2337 (1999)).

Plasmid DNA for use in the in vitro transformation of mammalian cells ormouse immunization experiments was purified by anion exchangechromatography as described in Example 1.

Example 10

Immunochemical characterization and evaluation of WNV prM and E proteinsexpressed by pCBWN. WNV specific gene products encoded by the pCBWNplasmid were expressed in COS-1 cells. Cells were electroporated andtransformed with pCBWN plasmid according to Chang et al. (J. Virol. 74:4244-4252 (2000)). Electroporated cells were seeded onto 75 cm cultureflasks or a 12-well tissue culture dish containing one sterilecoverslip/well. All flasks and 12-well plates were kept at 37° C., 5%CO₂ incubator. Forty hours following electroporation, coverslipscontaining adherent cells were removed from the wells, washed brieflywith PBS, fixed with acetone for 2 minutes at room temperature, andallowed to air dry.

Protein expression was detected using indirect immunofluorescenceantibody assay (IFA), as described in Example 2. Flavivirus E-proteinspecific monoclonal antibody (Mab) 4G2, WNV mouse hyperimmune asciticfluid (HIAF) and normal mouse serum (NMS) at 1:200 dilution in PBS wereused as the primary antibody to detect protein expression (Henchal etal., Am. J. Trop. Med. Hyg. 31: 830-836 (1982)).

Tissue culture medium was harvested 40 and 80 hours followingelectroporation. Antigen-capture (Ag-capture) ELISA was used to detectsecreted WN virus antigen in the culture medium of transientlytransformed COS-1 cells. The Mab 4G2 and horseradishperoxidase-conjugated Mab 6B6C-1 were used to capture the WN virusantigens and detect captured antigen, respectively (Chang et al., J.Virol. 74: 4244-4452 (2000); Henchal et al., Am. J. Trop. Med. Hyg. 31:830-836 (1983); Roehrig et al., Virology 128: 118-126 (1983)).

WN virus antigen in the medium was concentrated by precipitation with10% polyethylene glycol (PEG)-8000. The precipitant was resuspended inTNE buffer (50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 7.5), clarified bycentrifugation, and stored at 4° C. Alternatively, the precipitant wasresuspended in a lyophilization buffer (0.1 M TRIZMA and 0.4% bovineserum albumin in borate saline buffer, pH 9.0), lyophilized and storedat 4° C. Lyophilized preparations were used as antigen for theevaluation in MAC- and indirect IgG ELISAs.

WN virus-specific protein was detected by IFA on the transientlytransformed COS-1 cells. E, prM and M proteins expressed in these cellswere secreted into the culture medium. WN virus antigen concentrated byPEG precipitation was extracted with 7.0% ethanol to remove residual PEG(Aizawa et al., Appl. Enviro. Micro. 39: 54-57 (1980)). Ethanolextracted antigens and gradient-purified WN virions were analyzed on aNuPAGE, 4-12% gradient Bis-Tris Gel in a Excel Plus ElectrophoresisApparatus (Invitrogen Corp., Carlsbad, Calif.) and followed byelectroblotting onto nitrocellulose membranes using a Excel Plus BlotUnit (Invitrogen Corp.). WN virus-specific proteins produced by thetransiently transformed COS-1 cells were detected by WN virus specificmouse HIAF or flavivirus E protein reactive Mab 4G2 in a Western blotanalysis, using NMS as a negative serum control. The proteins displayedsimilar reactivity and identical molecular weights to the correspondinggradient purified virion E, prM and M protein derived from WN virusinfected suckling mouse brain (SMB).

In analysis of the NRA as an antigen for diagnostic ELISA, one vial oflyophilized NRA, representing antigen harvested from 40 ml of tissueculture fluid, was reconstituted in 1.0 ml of distilled water andcompared with the reconstituted WN virus infected suckling mouse brain(SMB) antigen provided as lyophilized as β-propiolactone-inactivatedsucrose-acetone extracts (Clarke et al., Am. J. Trop. Med. Hyg. 7:561-573 (1958)). All recombinant proteins, prM, M and E, had a similarreactivity to that of the gradient-purified virion E, prM and Mproteins.

Coded human specimens were tested concurrently with antigens in the sametest at the developmental stage. The MAC- and IgG ELISA protocolsemployed were identical to the published methods (Johnson et al., J.Clin. Microbiol. 38: 1827-1831 (2000); Martin et al., J. Clin.Microbiol. 38: 1823-1826. (2000)). Human serum specimens were obtainedfrom the serum bank in our facility, which consists of specimens sent tothe DVBID for WN virus confirmation testing during the 1999 outbreak Inthese tests, a screening MAC- and IgG ELISA were performed on a 1:400specimen dilution. Specimens yielding positive/negative (P/N) OD ratiosbetween 2 and 3 were considered suspect positives. Suspect serumspecimens were subject to confirmation as positives by both ELISAend-point titration and plaque-reduction neutralization test (PRNT). Allspecimens yielding P/N OD ratios greater than 3.0 were consideredpositives without further confirmatory testing.

An Ag-capture ELISA employing flavivirus-group reactive, anti-E Mab, 4G2and 6B6C-1, was used to detect NRA secreted into culture fluid of pCBWNtransformed COS-1 cells. The antigen could be detected in the medium oneday following transformation; and the maximum ELISA titer (1:32-1:64) inthe culture fluid without further concentration was observed between daytwo and day four. NRA was concentrated by PEG precipitation, resuspendedin a lyophilization buffer, and lyophilized for preservation. Fordiagnostic test development, one vial of lyophilized NRA wasreconstituted with 1.0 ml distilled water and titrated in the MAC- orindirect IgG ELISA using WN virus positive and negative reference humansera (Johnson et al., J. Clin. Microbiol. 38: 1827-1831 (2000); Martinetal., J. Clin. Microbiol. 38: 1823-1826 (2000)). Dilutions 1:320 and1:160 of the NRA were found to be the optimal concentrations for use inMAC- and IgG ELISA, respectively. These dilutions resulted in a P/NOD₄₅₀ ratio of 4.19 and 4.54, respectively, for MAC- and IgG test. TheWN virus SMB antigens produced by NY-6480 and Eg101 strains were used at1:320 and 1:640 dilution for MAC-ELISA, and 1:120 and 1:320 for IgGELISA, respectively. The negative control antigens, PEG precipitates ofthe culture medium of normal COS-1 cells and normal SMB antigen, wereused at the same dilutions as for the respective NRA and SMB antigen.Human serum specimens, diluted at 1:400, were tested concurrently intriplicate with virus-specific and negative control antigens. For thepositive test result to be valid, the OD₄₅₀ for the test serum reactedwith viral antigen (P) had to be at least two-fold greater than thecorresponding optical density value of the same serum reacted withnegative control antigen (N).

The reactivity of NRA and NY-06480, Eg101 and SLE virus SMBs werecompared by the MAC- and IgG ELISAs using 21 coded human serumspecimens. Of the 21 specimens, 19 had similar results on all threeantigens (8 negatives and 11 suspect positives or positives). Eighteenspecimens were also tested separately using SLE SMB antigen. Only threeof 13 Eg-101-SMB positive specimens were positive in the SLE MAC-ELISA(Table 1). None of WN antigen negative specimens was positive by SLEMAC-ELISA. This result confirmed a previous observation that anti-WNvirus IgM did not cross-react significantly with other flaviviruses(Tardei et al., J. Clin. Microbiol. 38: 2232-2239 (1940)) and wasspecific to diagnose acute WN virus infection regardless of whether NRAor SMB antigen was used in the test. All of the specimens were alsotested concurrently by indirect IgG ELISA. Ten of 21 specimens werepositive using any of the three antigens.

The two discrepant serum specimens (7 and 9) both from the same patient,collected on day-4 and 44 after onset of disease, respectively, wereIgM-negative with NRA and SMB NY antigen and IgM-positive using Eg-101SMB antigen in the initial test. To investigate these two discordantspecimens further, six sequentially collected specimens from thispatient were retested by end-point MAC- and IgG ELISAs. A greater than32-fold serial increase shown in the MAC-ELISA titer between day-3 andday-15 could be demonstrated with all antigens used. Cerebrospinal fluidcollected on day-9 after onset of disease also confirmed that thispatient indeed was infected by WN shortly prior to taking the sample.The cerebrospinal fluid had IgM P/N reading of 13.71 and 2.04 againstEg-101- and SLE-SMB antigens, respectively. Day-31 and day-44 specimenswere negative (<1:400) by using NY-SMB antigen but positive by using NRAand Eg101-SMB. Compatible IgG titers were observed with all threeantigen used in the test.

Example 11

Evaluation of the immune response in animals vaccinated with pCBWN.Groups of ten, three-wk-old female ICR mice were used in the study. Micewere injected intramuscularly (i.m.) with a single dose of pCBWN or agreen fluorescent protein expressing plasmid (PEGFP) DNA (Clonetech, SanFrancisco, Calif.). The pCBWN plasmid DNA was purified from XL-1 bluecells with EndoFree Plasmid Giga Kits (Qiagen) and resuspended in PBS,pH 7.5, at a concentration of 1.0 μg/μl. Mice that received 100 μg ofpEGFP were used as unvaccinated controls. Mice were injected with thepCBWN plasmid at a dose of 100, 10, 1.0, or 0.1 μg in a volume of 100μl. Groups that received 10, 1.0, or 0.1 μg of pCBWN were vaccinated bythe electrotransfer mediated in vivo gene delivery protocol using theEMC-830 square wave electroporator (Genetronics Inc. San Diego, Calif.).The electrotransfer protocol was based on the method of Mir et al.,(Proc. Natl. Acad. Sci. USA 96: 4262-4267.(1999)). Immediately followingDNA injection, transcutaneous electric pulses were applied by twostainless steel plate electrodes, placed 4.5-5.5 mm apart, at each sideof the leg. Electrical contact with the leg skin was ensured bycompletely wetting the leg with PBS. Two sets of four pulses of 40volts/mm of 25 msec duration with a 200 msec interval between pulseswere applied. The polarity of the electrode was reversed between the setof pulses to enhance electrotransfer efficiency.

Mice were bled every 3 wks following injection. The WN virus specificantibody response was evaluated by Ag-capture ELISA and plaque reductionneutralization test (PRNT). Individual sera were tested by IgG-ELISA,and pooled sera from 10 mice of each group were assayed by PRNT. All themice vaccinated with pCBWN had IgG ELISA titers ranging from 1:640 to1:1280 three wks after vaccination. The pooled sera collected at threeand six wks had a Nt antibody titer of 1:80. None of the serum specimensfrom pEGFP control mice displayed any ELISA or Nt titer to WN virus.

To determine if the single i.m. vaccination of pCBWN could protect micefrom WN virus infection, mice were challenged with NY-6480 virus eitherby intraperitoneal injection or by exposure to the bite ofvirus-infected Culex mosquitoes. Half of the mouse groups werechallenged intraperitoneally (ip) at 6 wks post vaccination with 1,000LD₅₀ (1,025 PFU/100 μl) of NY99-6480 virus. The remaining mice were eachexposed to the bites of three Culex tritaeniorhynchus mosquitoes thathas been infected with NY99-6480 virus 7 days prior to the challengeexperiment. Mosquitoes were allowed to feed on mice until they werefully engorged. Mice were observed twice daily for three wks afterchallenge.

It was evident that the presence of Nt antibodies correlated withprotective immunity, since all mice immunized with WN virus DNA remainedhealthy after virus challenge while all control mice developed symptomsof CNS infection 4-6 days following virus challenge and died on anaverage of 6.9 and 7.4 days after intraperitoneal or infective mosquitochallenge, respectively. In the vaccinated group, the pooled seracollected three wks after virus challenge (9-wk post immunization) hadNt antibody titers of 1:640 or 1:320. Pooled vaccinated mouse serareacted only with E protein in the Western blot analysis.

Groups of ten mice were immunized with 10.0 to 0.1 μg of pCBWN peranimal by use of electrotransfer. All groups that received pCBWN werecompletely protected from virus challenge. At 6 wks after immunizationall groups of electrotransfer mice had Nt titer less than four-folddifferent than animals receiving 100 μg of pCBWN by conventional i.m.injection without electrotransfer. Both these results evidencingeffective immunization suggest that the electrotransfer protocolenhances the immunogenicity and protective efficacy of the DNA vaccineof the invention (when carried out as described in (Mir et al., Proc.Natl. Acad. Sci. USA. 96: 4262-4267.(1999)).

Mixed-bred mares and geldings of various ages used in this study wereshown to be WN virus and SLE virus antibody-negative by ELISA and PRNT.Four horses were injected i.m. with a single dose (1,000 μg/1,000 μl inPBS, pH 7.5) of pCBWN plasmid. Serum specimens were collected everyother day for 38 days prior to virus challenge, and the WN virusspecific antibody response was evaluated by MAC- or IgG ELISA and PRNT.

Two days prior to virus challenge, 12 horses (4 vaccinated and 8control) were relocated into a bio-safety level (BSL)-3 containmentbuilding at the Colorado State University. The eight unvaccinatedcontrol horses were the subset of a study that was designed toinvestigate WN virus induced pathogenesis in horses and the potential ofhorses to serve as amplifying hosts. Horses were each challenged by thebite of 14 or 15 Aedes albopictus mosquitoes that had been infected byNY99-6425 or BC787 virus 12 days prior to horse challenge. Mosquitoeswere allowed to feed on horses for a period of 10 min. Horses wereexamined for signs of disease twice daily. Body temperature wasrecorded, and serum specimens collected twice daily from days 0 (day ofinfection) to 10, then once daily through day 14. Pulse and respirationwere recorded daily after challenge. The collected serum samples weretested by plaque titration for detection of viremia, and by MAC- or IgGELISA and PRNT for antibody response.

No systemic or local reaction was observed in any vaccinated horse.Individual horse sera were tested by PRNT. Vaccinated horses developedNt antibody greater than or equal to 1:5 between days 14 and 31. Endpoint titers for vaccinated horses, #5, #6, #7, and #8, on day-37 (twodays prior to mosquito challenge) were 1:40, 1:5, 1:20, and 1:20,respectively. Horses vaccinated with the pCBWN plasmid remained healthyafter virus challenge. None of them developed a detectible viremia orfever from days 1 to 14. All unvaccinated control horses became infectedwith WN virus after exposure to infected mosquito bites. Seven of theeight unvaccinated horses developed viremia that appeared during thefirst 6 days after virus challenge. Viremic horses developed Nt antibodybetween day-7 and day-9 after virus challenge. The only horse from theentire study to display clinical signs of disease was horse #11, whichbecame febrile and showed neurologic signs beginning 8 days afterinfection. This horse progressed to severe clinical disease within 24hours and was euthanized on day 9. Four representing horses, #9, 10, 14and 15, presenting viremia for 0, 2, 4, or 6 days, were selected andused as examples in this example. Virus titers ranged from 10^(1.0)PFU/ml of serum (in horse #10), the lowest level detectable in ourassay, to 10^(2.4)/ml (in horse #9). Horse #14 did not develop adetectible viremia during the test period. However, this horse wasinfected by the virus, as evidenced by Nt antibody detected after day12.

Anamnestic Nt antibody response was not observed in vaccinated horses asevidenced by the gradual increase in Nt titer during the experiment.Pre-existing Nt antibody in the vaccinated horse prior to mosquitochallenge could suppress initial virus infection and replication.Without virus replication, the challenge virus antigen provided byinfected mosquitoes may not contain a sufficient antigen mass tostimulate anamnestic immune response in the vaccinated horse. Allvaccinated horses were euthanized at 14 days after virus challenge.Gross pathological and histopathological lesions indicative of WN viralinfection were not observed.

Example 12

Preparation of recombinant plasmids containing coding sequences foryellow fever virus (YFV) or St. Louis encephalitis virus (SLEV) prM andE proteins. A strategy similar to constructing the pCDJE2-7 recombinantplasmid was used to prepare YFV and SLEV recombinant plasmids. GenomicRNA was extracted from 150 μL of YFV strain TRI-788379 or SLE strain78V-6507 virus seeds using Q1Aamp™ Viral RNA Kit (Qiagen, Santa Clarita,Calif.). The viral RNA was used as a template for amplification of YFVor SLEV prM and E gene coding regions. Primers YFDV389 (nucleotidesequence, SEQ ID NO:4; amino acid sequence, SEQ ID NO:5), cYFDV2452 (SEQID NO:6), SLEDV410 (nucleotide sequence, SEQ ID NO:7; amino acidsequence, SEQ ID NO: 8) and cSLEDV2449 (SEQ ID NO:9) were used togenerate the corresponding recombinant nucleic acids as described abovefor the preparation of the JEV and WNV recombinant plasmids. RT-PCRamplified cDNA, digested with KpnI and NotI enzymes, was inserted intothe KpnI-NotI site of a eukaryotic expression plasmid vector, pCDNA3(Invitrogen). Both strands of the cDNA were sequenced and verified foridentity to sequences from YFV strain TRI-788379 or SLEV strain78V-6507. Recombinant plasmids pCDYF2 and pCDSLE4-3, which contained thenucleotide sequences of the prM and E coding regions for YFV or SLEV,respectively, were purified using an EndoFree™ Plasmid Maxi Kit(Qiagen), and used for in vitro transformation or mouse immunization.

YFV or SLEV specific antigens were expressed in COS-1 cells transformedby pCDYF2 or pCDSLE4-3, respectively. The level of expressed proteinswas similar to a YFV- or SLEV-infected COS-1 cell control. As in the JEVmodel, COS-1 cell lines transformed by vectors bearing genes for theviral antigens were obtained which constitutively express YFV or SLEVantigenic proteins. Epitope mapping by IFA using a panel of YFV or SLEVE-specific Mabs indicated that the authentic E protein was expressed bythe pCDYF2- or pCDSLE4-3-transformed COS-1 cells. A preliminary studyindicated that 100% of three week-old female, ICR mice seroconvertedafter intramuscular inoculation with a single dose of 100 μg/100 μL ofpCDSLE4-3 plasmid in deionized water.

Example 13

Preparation of recombinant plasmids containing coding sequences for St.Louis encephalitis virus prM and E antigens with JEV signal sequence.Genomic RNA was extracted from 150 μL of Vero cell culture mediuminfected with MSI-7 strain of St. Louis encephalitis virus using theQIAamp™ Viral RNA Kit (Qiagen, Santa Clarita, Calif.). Extracted RNA waseluted and suspended in 80 μl of nuclease-free water, and used as atemplate for the amplification of St. Louis encephalitis virus prM and Egene coding sequences. Primer sequences were obtained from the work ofTrent et al. (Virology 156: 293-304 (1987)). A cDNA fragment containingthe genomic nucleotide region was amplified by the reversetranscriptase-polymerase chain reaction (RT-PCR). Restriction site AfeIwas engineered at the 5′ terminus of the cDNA by using amplimer SLE463(SEQ ID NO:30). An in-frame translation termination codon, followed by aNotI restriction site was introduced at the 3′ terminus of the cDNA byusing amplimer cSLE2447 (SEQ ID NO:31). The RT-PCR product was purifiedby a QIAquick™ PCR Purification Kit (Qiagen).

The double-stranded amplicon, produced by use of the two amplimers above(SEQ ID NO:30 and SEQ ID NO:31), was digested with AfeI and NotI enzymesto generate a 2004 fragment of DNA (463 to 2466 nt), and inserted intothe AfeI and NotI sites of a pCBJESS-M vector to form pCBSLE (nucleotidesequence, SEQ ID NO:21; amino acid sequence, SEQ ID NO:22). ThepCBJESS-M was derived from the pCBamp plasmid, that contained thecytomegalovirus early gene promoter and translational control elementand an engineered, modified JE signal sequence element (SEQ ID NO:27).The JE signal sequence element comprises the modified JE signal sequenceat −4 (Cys to Gly) and −2 (Gly to Ser) position in the original pCBJESSplasmid.

Automated DNA sequencing using an ABI prism 377 Sequencer (AppliedBiosystems/Perkin Elmer, Foster City, Calif.) was used to confirm thatthe recombinant plasmid had a correct prM and E sequence as defined byTrent et al. (Virology 156: 293-304 (1987)).

Example 14

Preparation of recombinant plasmids containing coding sequences foryellow fever virus (YFV) prM and E proteins with JEV signal sequence.Genomic RNA was extracted from 150 μL of Vero cell culture mediuminfected with 17D-213 strain of yellow fever virus using the QIAamp™Viral RNA Kit (Qiagen, Santa Clarita, Calif.). Extracted RNA was elutedand suspended in 80 μl of nuclease-free water, and used as a templatefor the amplification of yellow fever virus prM and E gene codingsequences. Primer sequences were obtained from the work of dos Santos etal. (Virus Research 35: 35-41 (1995)). A cDNA fragment containing thegenomic nucleotide region was amplified by the reversetranscriptase-polymerase chain reaction (RT-PCR). Restriction site AfeIwas engineered at the 5′ terminus of the cDNA by using amplimer YF482(SEQ ID NO:28). An in-frame translation termination codon, followed by aNotI restriction site was introduced at the 3′ terminus of the cDNA byusing amplimer cYF2433 (SEQ ID NO:29). The RT-PCR product was purifiedby a QIAquick™ PCR Purification Kit (Qiagen).

The double-stranded amplicon, produced by use of the two amplimers above(SEQ ID NO:28 and SEQ ID NO:29), was digested with AfeI and NotI enzymesto generate a 1971 fragment of DNA (482 to 2452 nt), and inserted intothe AfeI and NotI sites of a pCBJESS-M vector to form pCBYF (nucleotidesequence, SEQ ID NO:23; amino acid sequence, SEQ ID NO:24). ThepCBJESS-M was derived from the pCBamp plasmid, that contained thecytomegalovirus early gene promoter and translational control elementand an engineered JE signal sequence element (SEQ ID NO:27). The JEsignal sequence element comprises the modified JE signal sequence at −4(Cys to Gly) and −2 (Gly to Ser) position of JESS in the pCBJESSplasmid.

Automated DNA sequencing using an ABI prism 377 Sequencer (AppliedBiosystems/Perkin Elmer, Foster City, Calif.) was used to confirm thatthe recombinant plasmid had a correct prM and E sequence as defined bydos Santos et al. (Virus Research 35: 35-41 (1995)).

Example 15

Preparation of recombinant plasmids containing coding sequences forPowassan virus prM and E antigens with JEV signal sequence. Genomic RNAwas extracted from 150 μL of Vero cell culture medium infected with LBstrain of Powassan virus using the QIAamp™ Viral RNA Kit (Qiagen, SantaClarita, Calif.). Extracted RNA was eluted and suspended in 80 μl ofnuclease-free water, and used as a template for the amplification ofPowassan virus prM and E gene coding sequences. Primer sequences wereobtained from the work of Mandl et al. (Virology 194: 173-184 (1993)). AcDNA fragment containing the genomic nucleotide region was amplified bythe reverse transcriptase-polymerase chain reaction (RT-PCR).Restriction site AfeI was engineered at the 5′ terminus of the cDNA byusing amplimer POW454 (SEQ ID NO:25). An in-frame translationtermination codon, followed by a NotI restriction site was introduced atthe 3′ terminus of the cDNA by using amplimer cPOW2417 (SEQ ID NO:26).The RT-PCR product was purified by a QIAquick™ PCR Purification Kit(Qiagen).

The double-stranded amplicon, produced by use of the two amplimers above(SEQ ID NO:25 and SEQ ID NO:26), was digested with AfeI and NotI enzymesto generate a 1983 bp fragment of DNA (454 to 2436 nt), and insertedinto the AfeI and NotI sites of a pCBJESS-M vector to form pCBPOW(nucleotide sequence, SEQ ID NO:19; amino acid sequence, SEQ ID NO:20).The pCBJESS-M was derived from the pCBamp plasmid, that contained thecytomegalovirus early gene promoter and translational control elementand an engineered JE signal sequence element (SEQ ID NO:27). The JEsignal sequence element comprises the modified JE signal sequence at −4(Cys to Gly) and −2 (Gly to Ser) position of JESS in the pCBJESSplasmid.

Automated DNA sequencing using an ABI prism 377 Sequencer (AppliedBiosystems/Perkin Elmer, Foster City, Calif.) was used to confirm thatthe recombinant plasmid had a correct prM and E sequence as defined byMandl et al. (Virology 194:173-184, (1993)).

Example 16

Preparation of plasmids containing coding sequences for dengue serotype2 structural proteins. Procedures such as those carried out for otherflaviviruses (see Examples 1, 9 and 12-15) are to be followed to preparevectors including nucleic acid TU's for dengue serotype 2 antigens.According to the examples, the amplimers used for construction of thevectors may be chosen to engineer the normal dengue virus signalsequence or they may be chosen so as to engineer a signal sequence fromanother flavivirus, such as a modified Japanese encephalitis virussignal sequence.

A plasmid containing the dengue serotype 2 gene region from prM to E isto be constructed. The dengue serotype 2 prM and E genes (Deubel et al.,Virology 155:365-377 (1986); Gruenberg et al., J. Gen. Virol. 69:1301-1398 (1988); Hahn et al., Virology 162:167-180 (1988)) are to beligated into a plasmid such as pCDNA3, and then excised and cloned intovectors such as pCBamp, pCEP4, pREP4, or pRc/RSV (supplied byInvitrogen, Carlsbad, Calif.) to enable expression. If necessary, adengue serotype 2 virus-specific sequence encoded in a cDNA sequence maybe amplified using a procedure such as the polymerase chain reaction(PCR). Alternatively, if the viral RNA is the source of the gene region,a DNA sequence may be amplified by a RT-PCR procedure. A DNA fragmentincluding an initiation codon at the 5′ end, and a termination codon atthe 3′ end is to be cloned into an expression vector at an appropriaterestriction nuclease-specific site, in such a way that thecytomegalovirus (CMV) immediate early (IE) promoter, an initiationcodon, and a terminator, are operably linked to the dengue serotype 2virus sequence.

Example 17

Vaccination of mice using a dengue serotype 2 DNA vaccine. The dengueserotype 2 nucleic TU vaccine encoding the gene region from prM to Eprepared in Example 16 is to be suspended in a suitable pharmaceuticalcarrier, such as water for injection or buffered physiological saline,and injected intramuscularly into groups of weanling mice. Controlgroups receive a comparable plasmid preparation lacking the dengueserotype 2 specific genes. The generation of dengue serotype 2-specificantibodies, and/or of dengue serotype 2-specific immune system cytotoxiccells, is to be assessed at fixed intervals thereafter, for example atweekly intervals. At about two to four months after administration ofthe nucleic acid TU vaccine, mice are to be challenged with dengueserotype 2 virus. Levels of viremia are to be assessed at appropriateintervals thereafter, such as every second day. Passive protection bymaternal antibody is to be assessed as indicated in Example 8.

TABLE 1 Transient expression of JE prM and E proteins by variousrecombinant plasmids in two transferred cell lines. IFAintensity/percentage Vector Recombinant of antigen-positive cells*Promotor Intron Poly (A) ORI Plasmid COS-1 COS-7 pCDNAS CMV No BGH SV40pCDJE2-7 3+/40  3+/35 pCBamp CMV No BGH No pCBJE1-14 3+/45 nd pC1BampCMV Yes BGH No PC1BJES14 3+/39 nd pCEP4 CMV No SV40 OriP pCEJE 2+/4 2+/3 pREP4 RSV No SV40 OriP pREJE 1+/3  1+/2 pRe/RSV RSV No BGH SV40pRCJE 1+/3  1+/3 pCDNAS CMV No BGH SV40 pCDNA3/CAT — — *Various celllines were transformed with pCDNA3/CAT (negative control), pCDJE2-7,pCBJE1-14, pC1BJES14, pCEJEm pREJE, or pRCJE, Cells ere trypsinized 48hours later and tested by an indirect immunofluorescent antibody assay(IFA) with JE virus-specific HIAF. Data are presented as the intensity(scale of 1+ to 4+) and the percentage of IFA positive cells. ThepCDNA3/CAT transformed cells were used as the negative control.

TABLE 2 Characterization of proteins expressed by a pCDJE2-7 stablytransformed clone (JE-4B) of COS-1 cells with JE virus-reactiveantibodies. Biological Activity of Mab Immunofluorescent intensity ofMab or Biological cells antiserum Specificity Function JEV infected 4BMab: MC3 JEV Specific 2+ 2+ 2F2 JEV Specific HI, N 4+ 4+ 112 JEVSpecific 4+ 4+ 503 JEV Specific N 4+ 3+ 109 Subgroup HI 2+ 1+ N.04Subgroup HI, N 3+ 4+ 201 Subgroup 1+ 1+ 203 Subgroup 4+ 3+ 204 Subgroup2+ 2+ 301 Subgroup HI 2+ 2+ 504 Flavivirus 4+ 4+ 6B6C-1 Flavivirus 2+ 2+3B4C-4 VEE — — H1AF: Anti-JEV 4+ 3+ Anti-WEE — — PBS — —

TABLE 3 Persistence of the immune response in mice immunized withpCDJE2-7 or JE-VEX vaccine. ELISA Titer (log₁₀) PRNT_(90%) Titer 3 wks 6wks 9 wks 23 wks 40 wks 60 wks* 3 wks 6 wks 9 wks 1× pCDJE2-7 2.6-3.23.8-5.0 3.8-4.4 >3.2 >3.2 2.4, 2.4, 3.8, 4.4 <20  20 40-160 2× pCDJE2-72.6-3.8 4.4 3.8-4.4 >3.2 >3.2 2.6, 3.8, 3.8 <20 20-40 40-160 2× JE-VAX2.6-3.8 4.4-5.0 3.8-5.6 >3.2 >3.2 <2, <2, <2, 4.4 <20 20-40 20-160 2×pCDNA3/CAT <2 <2   <2 ND ND <2 <20 <20 <20 Mice were inoculated with 1or 2, 100 μg/dose plasmid DNA, or ⅕ human dose of JE-VAX vaccine. Serawere collected for testing prior to the second immunization. *Individualserum titers.

TABLE 4 The age-dependent percent seropositive rate in mice followingvaccination with various JEV vaccines. 3-day old 3-week old 3 weeks PV 7weeks PV 3 weeks PV 7 weeks PV JE-VAX 0 0 100 100 pCDNA3/CAT 0 0 0 0pCDJE2-7 40 60 90 90 PC1BJES14 10 60 80 100 pCBJE1-14 80 100 100 100

TABLE 5 Protection from JEV challenge in 8 week old mice followingvaccination at 3 days old with various JEV vaccines. Pre-challenge JEVDays post-challenge survival rate (%) Vaccine seroconversion 6 7 8 9 21JE-VAX 0 100 100 60 40 40 pCDNA3/CAT 0 100 80 30 30 30 pCDJE2-7 60 100100 100 100 100 PC1BJES14 60 100 100 100 100 100 pCBJE1-14 100 100 100100 100 100

TABLE 6 Evaluation of the ability of maternal antibody from JEV-nucleicacid-vaccinated female mice to protect their pups from fatal JEVencephalitis. JEV challenged pups Vaccinated mother Challenge ageVaccine PRNT_(90%) (days) No. survival¹ ELISA² 1× pCDJE2-7 40 4 0/11 2×pCDJE2-7 80 4 12/12  12/12 2× JE-VAX 20 3 0/16 2× pCDNA-3/CAT <10 5 0/141× pCDJE2-7 20 15 5/11 5/5 2× pCDJE2-7 40 14 8/12 7/8 2× JE-VAX 80 135/5  5/5 2× pCDNA-3/CAT <10 14 0/14 Mice were inoculated intramuscularlywith 1 or 2, 100 μg dose of plasmid DNA, or subcutaneously with two, ⅕human dose of JE-VAX vaccine. Sera were collected 9 weekspost-vaccination for PRNT testing prior to mating with non-immune male.¹No Survivors/total for each litter. ²Number of JEVELISA-antibody-positive animals (titer ≧ 1:400)/No. of survivors; serawere collected for testing 12 weeks after challenge.

1. A method of detecting a flavivirus antibody in a sample, comprising:(a) contacting the sample with a flavivirus antigen, which flavivirusantigen is an immunogenic flavivirus antigen produced by expressing atranscriptional unit encoding a Japanese encephalitis virus (JEV) signalsequence and an immunogenic flavivirus antigen of a flavivirus otherthan JEV, wherein the transcriptional unit directs the synthesis of theimmunogenic flavivirus antigen, under conditions whereby anantigen/antibody complex can form; and (b) detecting an increase inantigen/antibody complex formation in the sample relative to a negativecontrol, thereby detecting a flavivirus antibody in the sample.
 2. Themethod of claim 1, wherein the sample is obtained from a subject.
 3. Themethod of claim 1, wherein the transcriptional unit encodes anengineered JEV signal sequence.
 4. The method of claim 3, wherein theengineered JEV signal sequence comprises SEQ ID NO:14.
 5. The method ofclaim 1, wherein the immunogenic flavivirus antigen of a flavivirusother than JEV is of a flavivirus selected from the group consisting ofyellow fever virus, dengue serotype 1 virus, dengue serotype 2 virus,dengue serotype 3 virus, dengue serotype 4 virus, St. Louis encephalitisvirus, Powassan virus and West Nile virus.
 6. The method of claim 1,wherein the immunogenic flavivirus antigen of a flavivirus other thanJEV is selected from the group consisting of an M protein of aflavivirus, an E protein of a flavivirus, both an M protein and an Eprotein of a flavivirus, a portion of an M protein of flavivirus, aportion of an E protein of a flavivirus and both a portion of an Mprotein of a flavivirus and a portion of an E protein of a flavivirus orany combination thereof.
 7. The method of claim 6, wherein theimmunogenic flavivirus antigen of a flavivirus other than JEV is boththe M protein and the E protein of a flavivirus.
 8. The method of claim1, wherein the transcriptional unit comprises a control sequencedisposed appropriately such that it operably controls the synthesis ofthe immunogeic flavivirus antigen.
 9. The method of claim 8, wherein thecontrol sequence is the cytomegalovirus immediate early promoter. 10.The method of claim 1, comprising a Kozak consensus sequence located ata translational start site for a polypeptide comprising the immunogenicflavivirus antigen encoded by the transcriptional unit.
 11. The methodof claim 1, wherein the transcriptional unit comprises a poly-Aterminator.
 12. The method of claim 1, wherein the sample is a fluidsample or a tissue sample.
 13. The method of claim 12, wherein the fluidsample comprises at least one of cerebrospinal fluid, blood, bile,plasma, serum, saliva, urine, sputum and mucus.
 14. The method of claim1, wherein the sample is contacted with the flavivirus antigen in animmunofluorescence assay (IFA), an enzyme linked immunosorbent assay(ELISA), an immunoblotting assay or a microagglutination assay.
 15. Themethod of claim 1, further comprising contacting the antigen/antibodycomplex with a monoclonal antibody specific for at least one flavivirus.16. The method of claim 1, wherein the flavivirus antigen is bound to asubstrate.
 17. The method of claim 1, wherein the sample is at leastpartially purified prior to contacting with the flavivirus antigen. 18.The method of claim 3, wherein the engineered JEV signal sequencecomprises SEQ ID NO:27.
 19. A method of detecting a flavivirus antibodyin a sample, comprising: (a) contacting the sample with a flavivirusantigen, which flavivirus antigen is an immunogenic flavivirus antigenproduced by expressing a transcriptional unit encoding an engineeredJapanese encephalitis virus (JEV) signal sequence comprising SEQ ID NO:14, and an immunogenic flavivirus antigen comprising an M protein and anE protein of a flavivirus selected from the group consisting of yellowfever virus, dengue serotype 1 virus, dengue serotype 2 virus, dengueserotype 3 virus, dengue serotype 4 virus, St. Louis encephalitis virus,Powassan virus and West Nile virus, wherein the transcriptional unitdirects the synthesis of the immunogenic flavivirus antigen, underconditions whereby an antigen/antibody complex can form; and (b)detecting an increase in antigen/antibody complex formation in thesample relative to a negative control, thereby detecting a flavivirusantibody in the sample.
 20. A method of detecting a flavivirus antibodyin a sample, comprising: (a) contacting the sample with a flavivirusantigen, which flavivirus antigen is an immunogenic flavivirus antigenproduced by expressing a transcriptional unit encoding an engineeredJapanese encephalitis virus (JEV) signal sequence comprising SEQ ID NO:27, and an immunogenic flavivirus antigen comprising an M protein and anE protein of a flavivirus selected from the group consisting of yellowfever virus, dengue serotype 1 virus, dengue serotype 2 virus, dengueserotype 3 virus, dengue serotype 4 virus, St. Louis encephalitis virus,Powassan virus and West Nile virus, wherein the transcriptional unitdirects the synthesis of the immunogenic flavivirus antigen, underconditions whereby an antigen/antibody complex can form; and (b)detecting an increase in antigen/antibody complex formation in thesample relative to a negative control, thereby detecting a flavivirusantibody in the sample.